Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels.

PubMed

Barghaan, Jan; Tozakidou, Magdalini; Ehmke, Heimo; Bähring, Robert

2008-02-15

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2 Delta 2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2 Delta 2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

Piezoresistive cantilever force-clamp system

PubMed Central

Park, Sung-Jin; Petzold, Bryan C.; Goodman, Miriam B.; Pruitt, Beth L.

2011-01-01

We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or “clamps” the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of μN force and nm up to tens of μm displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode. PMID:21529009

Bromodomain and Extra-terminal (BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth.

PubMed

Niu, Ningning; Shao, Rui; Yan, Guang; Zou, Weiguo

2016-12-23

Small molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fabrication and characterization of GaN nanowire doubly clamped resonators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maliakkal, Carina B., E-mail: carina@tifr.res.in; Mathew, John P.; Hatui, Nirupam

2015-09-21

Gallium nitride (GaN) nanowires (NWs) have been intensely researched as building blocks for nanoscale electronic and photonic device applications; however, the mechanical properties of GaN nanostructures have not been explored in detail. The rigidity, thermal stability, and piezoelectric properties of GaN make it an interesting candidate for nano-electromechanical systems. We have fabricated doubly clamped GaN NW electromechanical resonators on sapphire using electron beam lithography and estimated the Young's modulus of GaN from resonance frequency measurements. For wires of triangular cross section with side ∼90 nm, we obtained values for the Young's modulus to be about 218 and 691 GPa, which are ofmore » the same order of magnitude as the values reported for bulk GaN. We also discuss the role of residual strain in the nanowire on the resonant frequency and the orientation dependence of the Young's modulus in wurtzite crystals.« less

Saddle clamp assembly

NASA Technical Reports Server (NTRS)

Belrose, Charles R. (Inventor)

1994-01-01

A saddle clamp assembly is presented. The assembly is comprised of a hollow cylindrical body centered about a longitudinal axis and being diametrically split into semicircular top and bottom sections. Each section has a pair of connection flanges, at opposite ends, that project radially outward. A pair of bolts are retained on the top section flanges and are threadable into nuts retained on the bottom section flanges. A base member is anchored to a central underside portion of the bottom clamp body section and has a pair of connection tabs positioned beneath the bottom clamp body section connection flanges on opposite sides of the clamp axis. A pair of bolts are retained on the base member connection tabs and are threadable into a pair of nuts retainable on a support structure. The connection tab and connection flanges on each side of the clamp body are axially offset in a manner permitting downward installation/removable tool access to the lower bolts past the connection flanges. An elongated retention tether is used to connect the top clamp body section to the balance of the clamp assembly. This prevents loss of the top clamp body section when it is removed from the bottom clamp body section.

Photovoltaic panel clamp

DOEpatents

Mittan, Margaret Birmingham [Oakland, CA; Miros, Robert H. J. [Fairfax, CA; Brown, Malcolm P [San Francisco, CA; Stancel, Robert [Loss Altos Hills, CA

2012-06-05

A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

Photovoltaic panel clamp

DOEpatents

Brown, Malcolm P.; Mittan, Margaret Birmingham; Miros, Robert H. J.; Stancel, Robert

2013-03-19

A photovoltaic panel clamp includes an upper and lower section. The interface between the assembled clamp halves and the module edge is filled by a flexible gasket material, such as EPDM rubber. The gasket preferably has small, finger like protrusions that allow for easy insertion onto the module edge while being reversed makes it more difficult to remove them from the module once installed. The clamp includes mounting posts or an integral axle to engage a bracket. The clamp also may include a locking tongue to secure the clamp to a bracket.

Selective Arterial Clamping Versus Hilar Clamping for Minimally Invasive Partial Nephrectomy.

PubMed

Yezdani, Mona; Yu, Sue-Jean; Lee, David I

2016-05-01

Partial nephrectomy has become an accepted treatment of cT1 renal masses as it provides improved long-term renal function compared to radical nephrectomy (Campbell et al. J Urol. 182:1271-9, 2009). Hilar clamping is utilized to help reduce bleeding and improve visibility during tumor resection. However, concern over risk of kidney injury with hilar clamping has led to new techniques to reduce length of warm ischemia time (WIT) during partial nephrectomy. These techniques have progressed over the years starting with early hilar unclamping, controlled hypotension during tumor resection, selective arterial clamping, minimal margin techniques, and off-clamp procedures. Selective arterial clamping has progressed significantly over the years. The main question is what are the exact short- and long-term renal effects from increasing clamp time. Moreover, does it make sense to perform these more time-consuming or more complex procedures if there is no long-term preservation of kidney function? More recent studies have shown no difference in renal function 6 months from surgery when selective arterial clamping or even hilar clamping is employed, although there is short-term improved decline in estimated glomerular filtration rate (eGFR) with selective clamping and off-clamp techniques (Komninos et al. BJU Int. 115:921-8, 2015; Shah et al. 117:293-9, 2015; Kallingal et al. BJU Int. doi: 10.1111/bju.13192, 2015). This paper reviews the progression of total hilar clamping to selective arterial clamping (SAC) and the possible difference its use makes on long-term renal function. SAC may be attempted based on surgeon's decision-making, but may be best used for more complex, larger, more central or hilar tumors and in patients who have renal insufficiency at baseline or a solitary kidney.

Rigid clamp

DOEpatents

Benavides, G.L.; Burt, J.D.

1994-07-12

The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object. 12 figs.

Rigid clamp

DOEpatents

Benavides, Gilbert L.; Burt, Jack D.

1994-01-01

The invention relates to a clamp mechanism that can be used to attach or temporarily support objects inside of tubular goods. The clamp mechanism can also be modified so that it grips objects. The clamp has a self-centering feature to accommodate out-of-roundness or other internal defections in tubular objects such as pipe. A plurality of clamping shoes are expanded by a linkage which is preferably powered by a motor to contact the inside of a pipe. The motion can be reversed and jaw elements can be connected to the linkage so as to bring the jaws together to grab an object.

Structural analysis of a eukaryotic sliding DNA clamp-clamp loadercomplex.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bowman, Gregory D.; O'Donnell, Mike; Kuriyan, John

2006-06-17

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-?-S by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA doublemore » helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.« less

Clamp-mount device

NASA Technical Reports Server (NTRS)

Clark, K. H. (Inventor)

1983-01-01

A clamp-mount device is disclosed for mounting equipment to an associated I-beam and the like structural member of the type having oppositely extending flanges wherein the device comprises a base and a pair of oppositely facing clamping members carried diagonally on the base clamping flanges therebetween and having flange receiving openings facing one another. Lock means are carried diagonally by the base opposite the clamping members locking the flanges in the clamping members. A resilient hub is carried centrally of the base engaging and biasing a back side of the flanges maintaining tightly clamped and facilitating use on vertical as well as horizontal members. The base turns about the hub to receive the flanges within the clamping members. Equipment may be secured to the base by any suitable means such as bolts in openings. Slidable gate latches secure the hinged locks in an upright locking position. The resilient hub includes a recess opening formed in the base and a rubber-like pad carried in this opening being depressably and rotatably carried therein.

Force-Measuring Clamp

NASA Technical Reports Server (NTRS)

Nunnelee, Mark (Inventor)

2004-01-01

A precision clamp that accurately measures force over a wide range of conditions is described. Using a full bridge or other strain gage configuration. the elastic deformation of the clamp is measured or detected by the strain gages. Thc strain gages transmit a signal that corresponds to the degree of stress upon the clamp. Thc strain gage signal is converted to a numeric display. Calibration is achieved by ero and span potentiometers which enable accurate measurements by the force-measuring clamp.

Mechanism of opening a sliding clamp

PubMed Central

Douma, Lauren G.; Yu, Kevin K.; England, Jennifer K.

2017-01-01

Abstract Clamp loaders load ring-shaped sliding clamps onto DNA where the clamps serve as processivity factors for DNA polymerases. In the first stage of clamp loading, clamp loaders bind and stabilize clamps in an open conformation, and in the second stage, clamp loaders place the open clamps around DNA so that the clamps encircle DNA. Here, the mechanism of the initial clamp opening stage is investigated. Mutations were introduced into the Escherichia coli β-sliding clamp that destabilize the dimer interface to determine whether the formation of an open clamp loader–clamp complex is dependent on spontaneous clamp opening events. In other work, we showed that mutation of a positively charged Arg residue at the β-dimer interface and high NaCl concentrations destabilize the clamp, but neither facilitates the formation of an open clamp loader–clamp complex in experiments presented here. Clamp opening reactions could be fit to a minimal three-step ‘bind-open-lock’ model in which the clamp loader binds a closed clamp, the clamp opens, and subsequent conformational rearrangements ‘lock’ the clamp loader–clamp complex in a stable open conformation. Our results support a model in which the E. coli clamp loader actively opens the β-sliding clamp. PMID:28973453

Sperm Patch-Clamp

PubMed Central

Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

2014-01-01

Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

PubMed Central

Armstrong, Lucas C.; Kirsch, Glenn E.; Fedorov, Nikolai B.; Wu, Caiyun; Kuryshev, Yuri A.; Sewell, Abby L.; Liu, Zhiqi; Motter, Arianne L.; Leggett, Carmine S.; Orr, Michael S.

2017-01-01

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors. PMID:28298165

Genetic and environmental influences on restrained eating behavior

PubMed Central

Schur, Ellen; Noonan, Carolyn; Polivy, Janet; Goldberg, Jack; Buchwald, Dedra

2009-01-01

Objective We examined the relative contributions of genetic and environmental influences to restrained eating. Methods Restrained eating was assessed by the Restraint Scale in a survey mailed to all twins enrolled in the University of Washington Twin Registry. We used structural equation modeling to estimate genetic and non-genetic contributions to restrained eating. Results 1,196 monozygotic, 456 same-sex dizygotic twins, and 447 opposite-sex twins were included in analyses. Restraint Scale scores were more closely correlated in monozygotic twins (rmale = 0.55, rfemale = 0.55) than in same-sex dizygotic twins (rmale = 0.31, rfemale = 0.19). Based on structural equation modeling, the estimated heritability for restrained eating, adjusted for BMI and sex, was 43% (95% confidence interval 35–50%). There was little evidence for common environmental effects. Conclusion These results indicate an inherited component to restrained eating. Genes could influence restrained eating directly or through inherited mediators such as personality factors or tendencies to gain weight. PMID:19658171

Being restrained: a study of power and powerlessness.

PubMed

Johnson, M E

1998-01-01

Previous research about the use of restraints has mainly sought generalizations regarding (a) who is likely to be restrained, (b) the frequency of the use of restraints, and (c) the behavior that precipitates the application of restraining devices. Therefore, there has been a paucity of research that attempts to understand the impact of restraint on the restrained person. The purpose of this study was to understand the meaning of the experience of being restrained for 10 psychiatric patients who had been restrained in leather restraints. Participants were interviewed in unstructured interviews. The taped interviews were transcribed and analyzed by means of a modification of an eight-stage interpretive process, grounded in Heideggerian phenomenology. In this article the theme of power is reported and discussed.

N-terminal nesprin-2 variants regulate β-catenin signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp.

PubMed

Duchi, Diego; Mazumder, Abhishek; Malinen, Anssi M; Ebright, Richard H; Kapanidis, Achillefs N

2018-06-06

RNA polymerase (RNAP) contains a mobile structural module, the 'clamp,' that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1-1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.

Split-tapered joint clamping device

DOEpatents

Olsen, Max J.; Schwartz, Jr., John F.

1988-01-01

This invention relates to a clamping device for removably attaching a tool element to a bracket element wherein a bracket element is disposed in a groove in the tool and a clamping member is disposed in said groove and in engagement with a clamping face of the bracket and a wall of the groove and with the clamping member having pivot means engaging the bracket and about which the clamping member rotates.

Comparison of Thoracic Injury Risk in Frontal Car Crashes for Occupant Restrained without Belt Load Limiters and Those Restrained with 6 kN and 4 kN Belt Load Limiters.

PubMed

Foret-Bruno, J Y; Trosseille, X; Page, Y; Huère, J F; Le Coz, J Y; Bendjellal, F; Diboine, A; Phalempin, T; Villeforceix, D; Baudrit, P; Guillemot, H; Coltat, J C

2001-11-01

In France, as in other countries, accident research studies show that a large proportion of restrained occupants who sustain severe or fatal injuries are involved in frontal impacts (65% and 50%, respectively). In severe frontal impacts with restrained occupants and where intrusion is not preponderant, the oldest occupants very often sustain severe thoracic injuries due to the conventional seat belt. As we have been observing over the last years, we will expect in the coming years developments which include more solidly-built cars, as offset crash test procedures are widely used to evaluate the passive safety of production vehicles. The reduction of intrusion for the most severe frontal impacts, through optimization of car deformation, usually translates into an increase in restraint forces and hence thoracic injury risk with a conventional retractor seat belt for a given impact severity. It is, therefore essential to limit the restraint forces exerted by the seat belt on the thorax in order to reduce the number of road casualties. In order to address thoracic injury risk in frontal impact, Renault cars have been equipped with the Programmed Restraint System (PRS) since 1995. The PRS is a restraint system that combines belt load limitation and pyrotechnic belt pretension. In an initial design of the Programmed Restraint System (PRS1), the belt load limiter was a steel component designed to shear at a given shoulder force, namely 6 kN. It was mounted between the retractor and the lower anchorage point of the belt. The design of the PRS was modified in 1998 (PRS2), but the principle of load limitation was maintained. The threshold was decreased to 4 kN and this lower belt belt-force limiter has been combined with a specially designed airbag. This paper reports on 347 real-world frontal accidents where the EES (Equivalent Energy Speed) ranged from 35 to 75 km/h. One hundred and ninety-eight (198) of these accidents involved cars equipped with the 6 kN load limiter

Restrained eaters show altered brain response to food odor.

PubMed

Kemmotsu, Nobuko; Murphy, Claire

2006-02-28

Do restrained and unrestrained eaters differ in their brain response to food odor? We addressed this question by examining restrained eaters' brain response to food (chocolate) and non-food (geraniol, floral) odors, both when odor was attended to and when ignored. Using olfactory event-related potentials (OERPs), we found that restrained eaters and controls responded similarly to the non-food odor; however, unlike controls, restrained eaters showed no increase in brain response to the food odor when they focused attention on it. Rather, restrained eaters showed attenuated OERP amplitudes to the food odor in both attended and ignored conditions, suggesting that the brain's response to attended food odor was abnormally suppressed.

Radial wedge flange clamp

DOEpatents

Smith, Karl H.

2002-01-01

A radial wedge flange clamp comprising a pair of flanges each comprising a plurality of peripheral flat wedge facets having flat wedge surfaces and opposed and mating flat surfaces attached to or otherwise engaged with two elements to be joined and including a series of generally U-shaped wedge clamps each having flat wedge interior surfaces and engaging one pair of said peripheral flat wedge facets. Each of said generally U-shaped wedge clamps has in its opposing extremities apertures for the tangential insertion of bolts to apply uniform radial force to said wedge clamps when assembled about said wedge segments.

Effects of introducing a single charged residue into the phenylalanine clamp of multimeric anthrax protective antigen.

PubMed

Janowiak, Blythe E; Fischer, Audrey; Collier, R John

2010-03-12

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe(427) residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe(427) in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity >or=1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

Delayed clamping of the umbilical cord after delivery and implications for public cord blood banking.

PubMed

Allan, David S; Scrivens, Nicholas; Lawless, Tiffany; Mostert, Karen; Oppenheimer, Lawrence; Walker, Mark; Petraszko, Tanya; Elmoazzen, Heidi

2016-03-01

Public banking of umbilical cord blood units (CBUs) containing higher numbers of cells ensures timely engraftment after transplantation for increasing numbers of patients. Delayed clamping of the umbilical cord after birth may benefit some infants by preventing iron deficiency. Implications of delayed cord clamping for public cord blood banking remains unclear. CBUs collected by Canadian Blood Services at one collection site between November 1, 2014, and March 17, 2015, were analyzed. The delay in cord clamping after birth was timed and classified as "no delay," 20 to 60 seconds, more than 60 seconds, or more than 120 seconds. Of 367 collections, 100 reported no delay in clamping while clamping was delayed by 20 to 60 seconds (n = 69), more than 60 seconds (n = 98), or more than 120 seconds (n = 100) in the remaining cases. The mean volume and total nucleated cells (TNCs) in units with no delay in clamping were significantly greater than mean volumes for all categories of delayed clamping (Tukey's test, p < 0.05 for each comparison). The proportion of units with more than 1.5 × 10(9) TNCs was significantly reduced when clamping was delayed (p = 5.5 × 10(-8) ). The difference was most marked for cords that were clamped more than 120 seconds after delivery (6.2% compared with 39%). Delayed cord clamping greatly diminishes the volume and TNC count of units collected for a public cord blood bank. Creating an inventory of CBUs with high TNC content may take more time than expected. © 2015 AABB.

Use of the Satinsky clamp for hilar clamping during robotic partial nephrectomy: indications, technique, and multi-center outcomes.

PubMed

Abdullah, Newaj; Rahbar, Haider; Barod, Ravi; Dalela, Deepansh; Larson, Jeff; Johnson, Michael; Mass, Alon; Zargar, Homayoun; Kaouk, Jihad; Allaf, Mohamad; Bhayani, Sam; Stifelman, Michael; Rogers, Craig

2017-03-01

A Satinsky clamp may be a backup option for hilar clamping during robotic partial nephrectomy (RPN) if there are challenges with application of bulldog clamps, but there are potential safety concerns. We evaluate outcomes of RPN using Satinsky vs. bulldog clamps, and provide tips for safe use of the Satinsky as a backup option. Using a multi-center database, we identified 1073 patients who underwent RPN between 2006 and 2013, and had information available about method of hilar clamping (bulldog clamp vs. Satinsky clamp). Patient baseline characteristics, tumor features, and perioperative outcomes were compared between the Satinsky and bulldog clamp groups. A Satinsky clamp was used for hilar clamping in 94 (8.8 %) RPN cases, and bulldog clamps were used in 979 (91.2 %) cases. The use of a Satinsky clamp was associated with greater operative time (198 vs. 175 min, p < 0.001), estimated blood loss (EBL, 200 vs. 100 ml, p < 0.001), warm ischemia time (WIT, 20 vs. 19 min, p = 0.036), transfusion rate (12.8 vs. 4.8 %, p = 0.001), and hospital stay (3 vs. 2 days, p < 0.001). Tumor characteristics and number of renal vessels were similar between groups. There were six intraoperative complications in the Satinsky clamp group, but none were directly related to the Satinsky clamp. On multivariable analysis, the use of the Satinsky clamp was not associated with increase in intraoperative or Clavien ≥3 postoperative complications, positive surgical margin rate or percentage change in estimated glomerular filtration rate. A Satinsky clamp can be a backup option for hilar clamping during challenging RPN cases, but requires careful technique, and was rarely necessary.

Quick action clamp

NASA Technical Reports Server (NTRS)

Calco, Frank S. (Inventor)

1991-01-01

A quick release toggle clamp that utilizes a spring that requires a deliberate positive action for disengagement is presented. The clamp has a sliding bolt that provides a latching mechanism. The bolt is moved by a handle that tends to remain in an engaged position while under tension.

N-terminal RASSF family

PubMed Central

Underhill-Day, Nicholas; Hill, Victoria

2011-01-01

Epigenetic inactivation of tumor suppressor genes is a hallmark of cancer development. RASSF1A (Ras Association Domain Family 1 isoform A) tumor suppressor gene is one of the most frequently epigenetically inactivated genes in a wide range of adult and children's cancers and could be a useful molecular marker for cancer diagnosis and prognosis. RASSF1A has been shown to play a role in several biological pathways, including cell cycle control, apoptosis and microtubule dynamics. RASSF2, RASSF4, RASSF5 and RASSF6 are also epigenetically inactivated in cancer but have not been analyzed in as wide a range of malignancies as RASSF1A. Recently four new members of the RASSF family were identified these are termed N-Terminal RASSF genes (RASSF7–RASSF10). Molecular and biological analysis of these newer members has just begun. This review highlights what we currently know in respects to structural, functional and molecular properties of the N-Terminal RASSFs. PMID:21116130

Laser beam guard clamps

DOEpatents

Dickson, Richard K.

2010-09-07

A quick insert and release laser beam guard panel clamping apparatus having a base plate mountable on an optical table, a first jaw affixed to the base plate, and a spring-loaded second jaw slidably carried by the base plate to exert a clamping force. The first and second jaws each having a face acutely angled relative to the other face to form a V-shaped, open channel mouth, which enables wedge-action jaw separation by and subsequent clamping of a laser beam guard panel inserted through the open channel mouth. Preferably, the clamping apparatus also includes a support structure having an open slot aperture which is positioned over and parallel with the open channel mouth.

How restrained eaters perceive the amount they eat.

PubMed

Jansen, A

1996-09-01

The cognitive model of binge eating states that it is the awareness of a broken diet that disinhibits the restrained eater. It is, according to that model, the perception of having overeaten that triggers disinhibited eating. However, although the perception of the amount eaten plays a central role in cognitive restraint theory, it has never directly been tested how restrained subjects perceive the amount of food they eat. In the present studies, participants were given ad libitum access to large amounts of palatable food and both their perception of the amount eaten and their estimated caloric intake were compared with the amount they actually ate. The restrained participants in these studies ate more than the unrestrained participants. In the first and second studies, the restrained participants consumed 571 and 372 'forbidden' calories respectively, without having the feeling that they had eaten very much, let alone too much. Moreover in both studies, the restrained eaters underestimated their caloric intake, whereas unrestrained eaters estimated their caloric intake quite well. The potential implications of the present findings for the cognitive restraint model are discussed.

Internal V-Band Clamp

DOEpatents

Vaughn, Mark R.; Hafenrichter, Everett S.; Chapa, Agapito C.; Harris, Steven M.; Martinez, Marcus J.; Baty, Roy S.

2006-02-28

A system for clamping two tubular members together in an end-to-end relationship uses a split ring with a V-shaped outer rim that can engage a clamping surface on each member. The split ring has a relaxed closed state where the ends of the ring are adjacent and the outside diameter of the split ring is less than the minimum inside diameter of the members at their ends. The members are clamped when the split ring is spread into an elastically stretched position where the ring rim is pressed tightly against the interior surfaces of the members. Mechanisms are provided for removing the spreader so the split ring will return to the relaxed state, releasing the clamped members.

Lifting clamp positively grips structural shapes

NASA Technical Reports Server (NTRS)

Reinhardt, E. C.

1966-01-01

Welded steel clamps securely grip structural shapes of various sizes for crane operations. The clamp has adjustable clamping jaws and screw-operated internal v-jaws and provides greater safety than hoisting slings presently used. The structural member can be rotated in any manner, angle, or direction without being released by the clamp.

Color-dependent learning in restrained Africanized honey bees.

PubMed

Jernigan, C M; Roubik, D W; Wcislo, W T; Riveros, A J

2014-02-01

Associative color learning has been demonstrated to be very poor using restrained European honey bees unless the antennae are amputated. Consequently, our understanding of proximate mechanisms in visual information processing is handicapped. Here we test learning performance of Africanized honey bees under restrained conditions with visual and olfactory stimulation using the proboscis extension response (PER) protocol. Restrained individuals were trained to learn an association between a color stimulus and a sugar-water reward. We evaluated performance for 'absolute' learning (learned association between a stimulus and a reward) and 'discriminant' learning (discrimination between two stimuli). Restrained Africanized honey bees (AHBs) readily learned the association of color stimulus for both blue and green LED stimuli in absolute and discriminatory learning tasks within seven presentations, but not with violet as the rewarded color. Additionally, 24-h memory improved considerably during the discrimination task, compared with absolute association (15-55%). We found that antennal amputation was unnecessary and reduced performance in AHBs. Thus color learning can now be studied using the PER protocol with intact AHBs. This finding opens the way towards investigating visual and multimodal learning with application of neural techniques commonly used in restrained honey bees.

New force replica exchange method and protein folding pathways probed by force-clamp technique.

PubMed

Kouza, Maksim; Hu, Chin-Kun; Li, Mai Suan

2008-01-28

We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes

Structure and geometry of the Aksay restraining double bend along the Altyn Tagh Fault, northern Tibet, imaged using magnetotelluric method

NASA Astrophysics Data System (ADS)

Xiao, Qibin; Yu, Guo; Liu-Zeng, Jing; Oskin, Michael E.; Shao, Guihang

2017-05-01

Large restraining bends along active strike-slip faults locally enhance the accumulation of clamping tectonic normal stresses that may limit the size of major earthquakes. In such settings, uncertain fault geometry at depth limits understanding of how effectively a bend arrests earthquake ruptures. Here we demonstrate fault imaging within a major restraining bend along the Altyn Tagh Fault of western China using the magnetotelluric (MT) method. The new MT data were collected along two profiles across the Aksay restraining double bend, which is bounded by two subparallel strands of the Altyn Tagh Fault: Northern (NATF) and Southern (SATF). Both two-dimensional (2-D) and three-dimensional (3-D) inversion models show that the Aksay bend may be the center of a positive flower structure, imaged as a high-resistivity body extending to an 40 km depth and bounded by subvertical resistivity discontinuities corresponding to the NATF and SATF. In the western section of the Aksay bend, both the NATF and SATF show similar low-resistivity structure, whereas in the eastern part of the bend, the low-resistivity anomaly below the SATF is wider and more prominent than that below the NATF. This observation indicates that the SATF shear zone may be wider and host more fluid than the NATF, lending structural support to the contention that fault slip at depth is asymmetrically focused on the SATF, even though surface slip is focused on the NATF. A south dipping, low-resistivity interface branching upward from the SATF toward the NATF indicates a fault link between these strands at depth.

Restrained eating and self-esteem in premenopausal and postmenopausal women.

PubMed

Drobnjak, Suzana; Atsiz, Semra; Ditzen, Beate; Tuschen-Caffier, Brunna; Ehlert, Ulrike

2014-01-01

There has been limited research about disordered eating in middle-aged women, and to date, few data exist about restrained eating behavior in postmenopausal women. Therefore, the aim of this study was to examine eating behavior with a specific focus on menopause as an associated factor in restrained eating. Beyond this, we were interested in how postmenopausal status and self-esteem would interact to determine eating patterns in women in middle age. We conducted an online survey in women aged between 40 and 66. Eating behavior was assessed with the Eating Disorder Examination-Questionnaire (EDE-Q) in premenopausal (N = 318) and postmenopausal women (N = 250). All participants rated their self-esteem using the Rosenberg Self-Esteem Scale (RSE) and reported their weight, height, waist circumference, and hip circumference. 15.7% of all participants showed clinically meaningful scores on restrained eating. Postmenopausal women showed significantly higher scores on the EDE-Q subscale of restrained eating as compared to premenopausal women, but when controlling for body mass index, however, this finding was no longer significant. Further exploratory analyses suggest that particularly low or high self-esteem levels are associated with restrained eating. Self-esteem might serve as a mediator between menopausal status and restrained eating, however results of these additional analyses were inconsistent. Restrained eating may appear in middle-aged women. Particularly in postmenopausal women, restrained eating might be associated with lower and higher self-esteem.

Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels.

PubMed

Tan, Peter S; Perry, Matthew D; Ng, Chai Ann; Vandenberg, Jamie I; Hill, Adam P

2012-09-01

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.

Detachable clamps for minimal access surgery.

PubMed

Frank, T; Willetts, G J; Cuschieri, A

1995-01-01

A detachable clamp and applicator have been developed for use in minimal access surgical operations involving hollow visceral transection and anastomosis. The clamp has parallel jaws which ensure uniform distribution of the occlusive force. Following application on the bowel, the clamp is released from the applicator, thus freeing the access port. On completion of the anastomosis, the clamp is docked to the applicator, its jaws opened for release from the bowel and then closed prior to removal. The jaws of the clamp are kept closed by a pseudoelastic nickel-titanium (NiTi) alloy spring which imparts advantageous force characteristics when compared to stainless steel. The excellent holding and atraumatic characteristics of the detachable clamp have been confirmed by use in laparoscopic and thoracoscopic surgery on the gastrointestinal tract.

Automated patch clamp on mESC-derived cardiomyocytes for cardiotoxicity prediction.

PubMed

Stoelzle, Sonja; Haythornthwaite, Alison; Kettenhofen, Ralf; Kolossov, Eugen; Bohlen, Heribert; George, Michael; Brüggemann, Andrea; Fertig, Niels

2011-09-01

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro-generated stem cell-derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell-derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell-derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

49 CFR 1103.22 - Restraining clients from improprieties.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 8 2010-10-01 2010-10-01 false Restraining clients from improprieties. 1103.22... Practitioner's Duties and Responsibilities Toward A Client § 1103.22 Restraining clients from improprieties. A practitioner should see that his clients act with the same restraint that the practitioner himself uses...

Restrained Differential Growth: The Initiating Event of Adolescent Idiopathic Scoliosis?

PubMed

Crijns, Tom Joris; Stadhouder, Agnita; Smit, Theodoor Henri

2017-06-15

An experimental model study and a short review of literature. The purpose of this study was to explore a new hypothesis suggesting that the curvatures seen in adolescent idiopathic scoliosis (AIS) originate from restrained differential growth between the vertebral column and the surrounding musculo-ligamentary structures. Despite decades of research, there is no generally accepted theory on the physical origin of the severe spinal deformations seen in AIS. The prevailing theories tend to focus on left-right asymmetry, rotational instability, or the sagittal spinal profile in idiopathic scoliosis. We test our hypothesis with a physical model of the spine that simulates growth, counteracted by ligaments and muscles, modeled by tethers and springs. Growth of the spine is further restrained by an anterior band representing the thorax, the linea alba, and abdominal musculature. We also explore literature in search of molecular mechanisms that may induce differential growth. Differential growth in the restrained spine model first induces hypokyphosis and mild lateral bending of the thoracic spine, but then suddenly escalates into a scoliotic deformity, consistent with clinical observations of AIS. The band simulating the ventral structures of the body had a pivotal effect on sagittal curvature and the initiation of lateral bending and rotation. In literature, several molecular mechanisms were found that may explain the occurrence of differential growth between the spine and the musculo-ligamentary structures. While AIS is a three-dimensional deformation of the spine, it appears that restrained differential growth in the sagittal plane can result in lateral bending and rotation without a pre-existing left-right asymmetry. This supports the concept that AIS may result from a growth imbalance rather than a local anatomical defect. N/A.

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

DOE PAGES

Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

2015-11-05

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

Oxidative Folding and N-terminal Cyclization of Onconase+

PubMed Central

Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

2008-01-01

Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pozo-Yauner, Luis del, E-mail: ldelpozo@inmegen.gob.mx; Wall, Jonathan S.; González Andrade, Martín

2014-01-10

Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stabilitymore » and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.« less

The relevance of restrained eating behavior for circadian eating patterns in adolescents

PubMed Central

Alexy, Ute; Diederichs, Tanja; Buyken, Anette E.; Roßbach, Sarah

2018-01-01

Background Restrained Eating, i.e. the tendency to restrict dietary intake to control body-weight, often emerges during adolescence and may result in changes in circadian eating patterns. Objective The objective of the present investigation was to determine the cross-sectional relevance of restrained eating for characteristics of circadian eating pattern in adolescents and whether changes in restrained eating are accompanied by concurrent changes in circadian eating pattern over the course of adolescence. Methods Two questionnaires assessing restrained eating (Score 0–30) with parallel 3-day weighed dietary records from two different time points were available from 209 (♂:101, ♀:108) 11–18 year old adolescents of the DONALD study. Mixed linear regression models were used to analyze whether restrained eating was associated with eating occasion frequency, snack frequency and morning and evening energy intake [in % of daily energy intake, %E]. Linear regression models were used to examine whether changes in restrained eating were associated with changes in the mentioned variables. Results Among girls, greater restrained eating was cross-sectionally associated with higher morning energy intake (p = 0.03). Further, there was a tendency towards lower evening energy intake with higher levels of restrained eating for the whole sample (p = 0.06). No cross-sectional associations were found with eating occasion or snack frequency. Each one-point increase in restrained eating during adolescence was related to a concurrent decrease in eating occasion frequency by 0.04 (95% CI -0.08; -0.01, p = 0.02) and in evening energy intake by 0.36%E (95% CI -0.70; -0.03, p = 0.04). A tendency towards decreasing snack frequency with increasing restrained eating was observed (β = -0.03, 95% CI -0.07; 0.00, p = 0.07). No association was found between changes in restrained eating and concurrent changes in morning energy intake. Conclusion We found indications for cross-sectional and

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

PubMed Central

Gerrits, Bertran; Roschitzki, Bernd; Mohanty, Sonali; Niederer, Eva M.; Laczko, Endre; Timmerman, Evy; Lange, Vinzenz; Hafen, Ernst; Aebersold, Ruedi; Vandekerckhove, Joël; Basler, Konrad; Ahrens, Christian H.; Gevaert, Kris; Brunner, Erich

2009-01-01

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species. PMID:19885390

The use of clamping grips and friction pads by tree frogs for climbing curved surfaces

PubMed Central

Ji, Aihong; Yuan, Shanshan; Hill, Iain; Wang, Huan; Barnes, W. Jon P.; Dai, Zhendong; Sitti, Metin

2017-01-01

Most studies on the adhesive mechanisms of climbing animals have addressed attachment against flat surfaces, yet many animals can climb highly curved surfaces, like twigs and small branches. Here we investigated whether tree frogs use a clamping grip by recording the ground reaction forces on a cylindrical object with either a smooth or anti-adhesive, rough surface. Furthermore, we measured the contact area of fore and hindlimbs against differently sized transparent cylinders and the forces of individual pads and subarticular tubercles in restrained animals. Our study revealed that frogs use friction and normal forces of roughly a similar magnitude for holding on to cylindrical objects. When challenged with climbing a non-adhesive surface, the compressive forces between opposite legs nearly doubled, indicating a stronger clamping grip. In contrast to climbing flat surfaces, frogs increased the contact area on all limbs by engaging not just adhesive pads but also subarticular tubercles on curved surfaces. Our force measurements showed that tubercles can withstand larger shear stresses than pads. SEM images of tubercles revealed a similar structure to that of toe pads including the presence of nanopillars, though channels surrounding epithelial cells were less pronounced. The tubercles' smaller size, proximal location on the toes and shallow cells make them probably less prone to buckling and thus ideal for gripping curved surfaces. PMID:28228509

N-Terminal Acetylation Inhibits Protein Targeting to the Endoplasmic Reticulum

PubMed Central

Forte, Gabriella M. A.; Pool, Martin R.; Stirling, Colin J.

2011-01-01

Amino-terminal acetylation is probably the most common protein modification in eukaryotes with as many as 50%–80% of proteins reportedly altered in this way. Here we report a systematic analysis of the predicted N-terminal processing of cytosolic proteins versus those destined to be sorted to the secretory pathway. While cytosolic proteins were profoundly biased in favour of processing, we found an equal and opposite bias against such modification for secretory proteins. Mutations in secretory signal sequences that led to their acetylation resulted in mis-sorting to the cytosol in a manner that was dependent upon the N-terminal processing machinery. Hence N-terminal acetylation represents an early determining step in the cellular sorting of nascent polypeptides that appears to be conserved across a wide range of species. PMID:21655302

Inertial piezoelectric linear motor driven by a single-phase harmonic wave with automatic clamping mechanism

NASA Astrophysics Data System (ADS)

He, Liangguo; Chu, Yuheng; Hao, Sai; Zhao, Xiaoyong; Dong, Yuge; Wang, Yong

2018-05-01

A novel, single-phase, harmonic-driven, inertial piezoelectric linear motor using an automatic clamping mechanism was designed, fabricated, and tested to reduce the sliding friction and simplify the drive mechanism and power supply control of the inertial motor. A piezoelectric bimorph and a flexible hinge were connected in series to form the automatic clamping mechanism. The automatic clamping mechanism was used as the driving and clamping elements. A dynamic simulation by Simulink was performed to prove the feasibility of the motor. The finite element method software COMSOL was used to design the structure of the motor. An experimental setup was built to validate the working principle and evaluate the performance of the motor. The prototype motor outputted a no-load velocity of 3.178 mm/s at a voltage of 220 Vp-p and a maximum traction force of 4.25 N under a preload force of 8 N. The minimum resolution of 1.14 μm was achieved at a driving frequency of 74 Hz, a driving voltage of 50 Vp-p, and a preload force of 0 N.

The scorpion toxin Bot IX is a potent member of the α-like family and has a unique N-terminal sequence extension.

PubMed

Martin-Eauclaire, Marie-France; Salvatierra, Juan; Bosmans, Frank; Bougis, Pierre E

2016-09-01

We report the detailed chemical, immunological and pharmacological characterization of the α-toxin Bot IX from the Moroccan scorpion Buthus occitanus tunetanus venom. Bot IX, which consists of 70 amino acids, is a highly atypical toxin. It carries a unique N-terminal sequence extension and is highly lethal in mice. Voltage clamp recordings on oocytes expressing rat Nav1.2 or insect BgNav1 reveal that, similar to other α-like toxins, Bot IX inhibits fast inactivation of both variants. Moreover, Bot IX belongs to the same structural/immunological group as the α-like toxin Bot I. Remarkably, radioiodinated Bot IX competes efficiently with the classical α-toxin AaH II from Androctonus australis, and displays one of the highest affinities for Nav channels. © 2016 Federation of European Biochemical Societies.

Management of umbilical cord clamping.

PubMed

Webbon, Lucy

2013-02-01

The Royal College of Midwives (RCM) has updated its third stage of labour guidelines (RCM 2012) to be clearly supportive of a delay in umbilical cord clamping, although specific guidance on timing is yet to be announced. It is therefore imperative that both midwives and student midwives understand and are able to integrate delaying into their practice, as well as communicating to women the benefits; only in this way can we give women fully informed choices on this aspect of care. The main benefit of delayed cord clamping is the protection it can provide in reducing childhood anaemia, which is a major issue, especially in poorer countries. A review of the evidence found no risks linked to delayed clamping, and no evidence that it cannot be used in combination with the administration of uterotonic drugs. Delayed cord clamping can be especially beneficial for pre term and compromised babies.

Payload Launch Lock Mechanism

NASA Technical Reports Server (NTRS)

Young, Ken (Inventor); Hindle, Timothy (Inventor)

2014-01-01

A payload launch lock mechanism includes a base, a preload clamp, a fastener, and a shape memory alloy (SMA) actuator. The preload clamp is configured to releasibly restrain a payload. The fastener extends, along an axis, through the preload clamp and into the base, and supplies a force to the preload clamp sufficient to restrain the payload. The SMA actuator is disposed between the base and the clamp. The SMA actuator is adapted to receive electrical current and is configured, upon receipt of the electrical current, to supply a force that causes the fastener to elongate without fracturing. The preload clamp, in response to the fastener elongation, either rotates or pivots to thereby release the payload.

A clamped rectangular plate containing a crack

NASA Technical Reports Server (NTRS)

Tang, R.; Erdogan, F.

1985-01-01

The general problem of a rectangular plate clamped along two parallel sides and containing a crack parallel to the clamps is considered. The problem is formulated in terms of a system of singular integral equations and the asymptotic behavior of the stress state near the corners is investigated. Numerical examples are considered for a clamped plate without a crack and with a centrally located crack, and the stress intensity factors and the stresses along the clamps are calculated.

N-terminal acetylation modulates Bax targeting to mitochondria.

PubMed

Alves, Sara; Neiri, Leire; Chaves, Susana Rodrigues; Vieira, Selma; Trindade, Dário; Manon, Stephen; Dominguez, Veronica; Pintado, Belen; Jonckheere, Veronique; Van Damme, Petra; Silva, Rui Duarte; Aldabe, Rafael; Côrte-Real, Manuela

2018-02-01

The pro-apoptotic Bax protein is the main effector of mitochondrial permeabilization during apoptosis. Bax is controlled at several levels, including post-translational modifications such as phosphorylation and S-palmitoylation. However, little is known about the contribution of other protein modifications to Bax activity. Here, we used heterologous expression of human Bax in yeast to study the involvement of N-terminal acetylation by yNaa20p (yNatB) on Bax function. We found that human Bax is N-terminal (Nt-)acetylated by yNaa20p and that Nt-acetylation of Bax is essential to maintain Bax in an inactive conformation in the cytosol of yeast and Mouse Embryonic Fibroblast (MEF) cells. Bax accumulates in the mitochondria of yeast naa20Δ and Naa25 -/- MEF cells, but does not promote cytochrome c release, suggesting that an additional step is required for full activation of Bax. Altogether, our results show that Bax N-terminal acetylation by NatB is involved in its mitochondrial targeting. Copyright © 2017 Elsevier Ltd. All rights reserved.

Micromachined patch-clamp apparatus

DOEpatents

Okandan, Murat

2012-12-04

A micromachined patch-clamp apparatus is disclosed for holding one or more cells and providing electrical, chemical, or mechanical stimulation to the cells during analysis with the patch-clamp technique for studying ion channels in cell membranes. The apparatus formed on a silicon substrate utilizes a lower chamber formed from silicon nitride using surface micromachining and an upper chamber formed from a molded polymer material. An opening in a common wall between the chambers is used to trap and hold a cell for analysis using the patch-clamp technique with sensing electrodes on each side of the cell. Some embodiments of the present invention utilize one or more electrostatic actuators formed on the substrate to provide mechanical stimulation to the cell being analyzed, or to provide information about mechanical movement of the cell in response to electrical or chemical stimulation.

Cell-Detection Technique for Automated Patch Clamping

NASA Technical Reports Server (NTRS)

McDowell, Mark; Gray, Elizabeth

2008-01-01

A unique and customizable machinevision and image-data-processing technique has been developed for use in automated identification of cells that are optimal for patch clamping. [Patch clamping (in which patch electrodes are pressed against cell membranes) is an electrophysiological technique widely applied for the study of ion channels, and of membrane proteins that regulate the flow of ions across the membranes. Patch clamping is used in many biological research fields such as neurobiology, pharmacology, and molecular biology.] While there exist several hardware techniques for automated patch clamping of cells, very few of those techniques incorporate machine vision for locating cells that are ideal subjects for patch clamping. In contrast, the present technique is embodied in a machine-vision algorithm that, in practical application, enables the user to identify good and bad cells for patch clamping in an image captured by a charge-coupled-device (CCD) camera attached to a microscope, within a processing time of one second. Hence, the present technique can save time, thereby increasing efficiency and reducing cost. The present technique involves the utilization of cell-feature metrics to accurately make decisions on the degree to which individual cells are "good" or "bad" candidates for patch clamping. These metrics include position coordinates (x,y) in the image plane, major-axis length, minor-axis length, area, elongation, roundness, smoothness, angle of orientation, and degree of inclusion in the field of view. The present technique does not require any special hardware beyond commercially available, off-the-shelf patch-clamping hardware: A standard patchclamping microscope system with an attached CCD camera, a personal computer with an imagedata- processing board, and some experience in utilizing imagedata- processing software are all that are needed. A cell image is first captured by the microscope CCD camera and image-data-processing board, then the image

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth*

PubMed Central

Kelly, Geraldine M.; Buckley, Deirdre A.; Kiely, Patrick A.; Adams, David R.; O'Connor, Rosemary

2012-01-01

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the 1248SFYYS1252 motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling. PMID:22685298

Conformationally restrained aromatic analogues of fosmidomycin and FR900098.

PubMed

Kurz, Thomas; Schlüter, Katrin; Pein, Miriam; Behrendt, Christoph; Bergmann, Bärbel; Walter, Rolf D

2007-07-01

The synthesis and in-vitro antimalarial activity of conformationally restrained bis(pivaloyloxymethyl) ester analogues of the natural product fosmidomycin is presented. In contrast to alpha-aryl-substituted analogues, conformationally restrained aromatic analogues exhibit only moderate in-vitro antimalarial activity against the chloroquine-sensitive strain 3D7 of Plasmodium falciparum. The most active derivative displays an IC(50) value of 47 microM.

The N-terminal strand modulates immunoglobulin light chain fibrillogenesis.

PubMed

del Pozo-Yauner, Luis; Wall, Jonathan S; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L; Pérez Carreón, Julio I; Ochoa-Leyva, Adrián; Fernández-Velasco, D Alejandro

2014-01-10

It has been suggested that the N-terminal strand of the light chain variable domain (V(L)) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V(L) protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Clamp force and alignment checking device

DOEpatents

Spicer, John Patrick; Cai, Wayne W.; Chakraborty, Debejyo; Mink, Keith

2017-04-11

A check fixture measures a total clamp force applied by a welder device. The welder device includes a welding horn having a plurality of weld pads and welding anvil having a plurality of weld pads. The check fixture includes a base member operatively supporting a plurality of force sensors. The base member and the force sensors are received between the weld pads of the welding horn and the anvil pads of the welding anvil. Each force sensor is configured to measure an individual clamp force applied thereto by corresponding weld and anvil pads when the base member is received between the welding horn and the welding anvil and the welder device is in the clamped position. The individual clamp forces are used to determine whether the weld and/or anvil pads are worn or misaligned.

Advanced motor driven clamped borehole seismic receiver

DOEpatents

Engler, Bruce P.; Sleefe, Gerard E.; Striker, Richard P.

1993-01-01

A borehole seismic tool including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric meter in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Plastic Clamp Retains Clevis Pin

NASA Technical Reports Server (NTRS)

Cortes, R. G.

1983-01-01

Plastic clamp requires no special installation or removal tools. Clamp slips easily over end of pin. Once engaged in groove, holds pin securely. Installed and removed easily without special tools - screwdriver or putty knife adequate for prying out of groove. Used to retain bearings, rollers pulleys, other parts that rotate. Applications include slowly and intermittently rotating parts in appliances.

Timing of umbilical cord-clamping and infant anaemia: the role of maternal anaemia.

PubMed

Blouin, Brittany; Penny, Mary E; Maheu-Giroux, Mathieu; Casapía, Martín; Aguilar, Eder; Silva, Hermánn; Creed-Kanashiro, Hilary M; Joseph, Serene A; Gagnon, Anita; Rahme, Elham; Gyorkos, Theresa W

2013-05-01

Evidence from randomized controlled trials has shown that delayed cord-clamping is beneficial to infant iron status. The role of maternal anaemia in this relationship, however, has not been established. To determine the effect of maternal anaemia at delivery on the association between timing of umbilical cord-clamping and infant anaemia at 4 and 8 months of age. A cohort of pregnant women admitted to the labour room of Hospital Iquitos (Iquitos, Peru) and their newborns were recruited into the study during two time periods (18 May to 3 June and 6-20 July 2009). Between the two recruitment periods, the hospital's policy changed from early to delayed umbilical cord-clamping. Maternal haemoglobin levels were measured before delivery, and the time between delivery and cord-clamping was recorded at delivery for the entire cohort. Mother-infant pairs were followed-up at 4 (n = 207) and 8 months (n = 184) post partum. Infant haemoglobin levels were measured at follow-up visits. Data were analysed using logistic regression models. The prevalence of maternal anaemia (Hb <11.0 g/dl) at delivery was 22%. Infant haemoglobin levels at 4 and 8 months of age were 10.4 g/dl and 10.3 g/dl, respectively. Infant haemoglobin levels did not differ significantly between infants born to anaemic mothers and those born to non-anaemic mothers at either 4 or 8 months of age. However, the association between the timing of cord-clamping and infant anaemia was modified by the mother's anaemia status. Significant benefits of delayed cord-clamping in preventing anaemia were found in infants born to anaemic mothers at both 4 months (aOR = 0.59, 95% CI 0.36-0.99) and 8 months (aOR = 0.38, 95% CI 0.19-0.76) of age. The study contributes additional evidence in support of delayed cord-clamping. This intervention is likely to have most public health impact in areas with a high prevalence of anaemia during pregnancy.

Cable clamp bolt fixture facilitates assembly in close quarters

NASA Technical Reports Server (NTRS)

Sunderland, G. H.

1967-01-01

Cable clamp bolt holding fixture facilitates forming of electrical cable runs in limited equipment space. The fixture engages the threads of the short clamp bolt through the clamp and maintains tension against clamp tendency to open while the operator installs the nut without difficulty.

Urinary urea nitrogen excretion during the hyperinsulinemic euglycemic clamp in type 1 diabetic patients and healthy subjects.

PubMed

Wohl, P; Krusinová, E; Klementová, M; Wohl, P; Kratochvílová, S; Pelikánová, T

2008-01-01

The hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry (IC) is used for estimation of insulin-stimulated substrate utilization. Calculations are based on urinary urea nitrogen excretion (UE), which is influenced by correct urine collection. The aims of our study were to improve the timing of urine collection during the clamp and to test the effect of insulin on UE in patients with type 1 diabetes (DM1; n=11) and healthy subjects (C; n=11). Urine samples were collected (a) over 24 h divided into 3-h periods and (b) before and during two-step clamp (1 and 10 mIU.kg(-1).min(-1); period 1 and period 2) combined with IC. The UE during the clamp was corrected for changes in urea pool size (UEc). There were no significant differences in 24-h UE between C and DM1 and no circadian variation in UE in either group. During the clamp, serum urea decreased significantly in both groups (p<0.01). Therefore, UEc was significantly lower as compared to UE not adjusted for changes in urea pool size both in C (p<0.001) and DM1 (p<0.001). While UE did not change during the clamp, UEc decreased significantly in both groups (p<0.01). UEc during the clamp was significantly higher in DM1 compared to C both in period 1 (p<0.05) and period 2 (p<0.01). The UE over 24 h and UEc during the clamp were statistically different in both C and DM1. We conclude that urine collection performed during the clamp with UE adjusted for changes in urea pool size is the most suitable technique for measuring substrate utilization during the clamp both in DM1 and C. Urine collections during the clamp cannot be replaced either by 24-h sampling (periods I-VII) or by a single 24-h urine collection. Attenuated insulin-induced decrease in UEc in DM1 implicates the impaired insulin effect on proteolysis.

Advanced motor driven clamped borehole seismic receiver

DOEpatents

Engler, B.P.; Sleefe, G.E.; Striker, R.P.

1993-02-23

A borehole seismic tool is described including a borehole clamp which only moves perpendicular to the borehole. The clamp is driven by an electric motor, via a right angle drive. When used as a seismic receiver, the tool has a three part housing, two of which are hermetically sealed. Accelerometers or geophones are mounted in one hermetically sealed part, the electric motor in the other hermetically sealed part, and the clamp and right angle drive in the third part. Preferably the tool includes cable connectors at both ends. Optionally a shear plate can be added to the clamp to extend the range of the tool.

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

21 CFR 882.4460 - Neurosurgical head holder (skull clamp).

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Neurosurgical head holder (skull clamp). 882.4460... holder (skull clamp). (a) Identification. A neurosurgical head holder (skull clamp) is a device used to clamp the patient's skull to hold head and neck in a particular position during surgical procedures. (b...

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney

PubMed Central

DeSilva, Kristin; Sorice, Gian Pio; Muscogiuri, Giovanna; Jimenez, Fabio; Ahuja, Seema; Barnes, Jefferey L.; Choudhury, Goutam Ghosh; Musi, Nicolas; DeFronzo, Ralph; Kasinath, Balakuntalam S.

2013-01-01

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease. PMID:24108867

Combined acute hyperglycemic and hyperinsulinemic clamp induced profibrotic and proinflammatory responses in the kidney.

PubMed

Mariappan, Meenalakshmi M; DeSilva, Kristin; Sorice, Gian Pio; Muscogiuri, Giovanna; Jimenez, Fabio; Ahuja, Seema; Barnes, Jefferey L; Choudhury, Goutam Ghosh; Musi, Nicolas; DeFronzo, Ralph; Kasinath, Balakuntalam S

2014-02-01

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

Positive fantasies or negative contrasts: the effect of media body ideals on restrained eaters' mood, weight satisfaction, and food intake.

PubMed

Boyce, Jessica A; Kuijer, Roeline G; Gleaves, David H

2013-09-01

Although viewing media body ideals promotes body dissatisfaction and problematic eating among women (e.g., extreme restraint/overeating), some argue that women only report such negative effects because they think that they are meant to (i.e., demand characteristics). Because restrained eaters are trying to lose weight, they might be vulnerable to such media exposure. However, because of demand characteristics, evidence is mixed. Therefore, we minimized demand characteristics and explored whether media body ideals would trigger restrained eaters to report negative (negative mood, weight dissatisfaction) or positive (positive mood, weight satisfaction) effects. We also hypothesized that this change (negative or positive) would encourage food intake. Restrained and unrestrained eaters (n=107) memorized media or control images. Restrained eaters exposed to media images reported decreased weight satisfaction and increased negative mood, but their food intake was not significantly affected. Perhaps paying advertent attention to the images caused goal-related negative affect, which triggered restraint. Copyright © 2013 Elsevier Ltd. All rights reserved.

Patch-clamp amplifiers on a chip

PubMed Central

Weerakoon, Pujitha; Culurciello, Eugenio; Yang, Youshan; Santos-Sacchi, Joseph; Kindlmann, Peter J.; Sigworth, Fred J.

2010-01-01

We present the first, fully-integrated, two-channel implementation of a patch-clamp measurement system. With this “PatchChip” two simultaneous whole-cell recordings can be obtained with rms noise of 8 pA in a 10 kHz bandwidth. The capacitance and series-resistance of the electrode can be compensated up to 10 pF and 100 MΩ respectively under computer control. Recordings of hERG and Nav 1.7 currents demonstrate the system's capabilities, which are on par with large, commercial patch-clamp instrumentation. By reducing patch-clamp amplifiers to a millimeter size micro-chip, this work paves the way to the realization of massively-parallel, high-throughput patch-clamp systems for drug screening and ion-channel research. The PatchChip is implemented in a 0.5 μm silicon-on-sapphire process; its size is 3 × 3 mm2 and the power consumption is 5 mW per channel with a 3.3 V power supply. PMID:20637803

Combination Space Station Handrail Clamp and Pointing Device

NASA Technical Reports Server (NTRS)

Hughes, Stephen J. (Inventor)

1999-01-01

A device for attaching an experiment carrier to a space station handrail is provided. The device has two major components, a clamping mechanism for attachment to a space station handrail, and a pointing carrier on which an experiment package can be mounted and oriented. The handrail clamp uses an overcenter mechanism and the carrier mechanism uses an adjustable preload ball and socket for carrier positioning. The handrail clamp uses a stack of disk springs to provide a spring loaded button. This configuration provides consistent clamping force over a range of possible handrail thicknesses. Three load points are incorporated in the clamping mechanism thereby spreading the clamping load onto three separate points on the handrail. A four bar linkage is used to provide for a single actuation lever for all three load points. For additional safety, a secondary lock consisting of a capture plate and push lock keeps the clamp attached to the handrail in the event of main clamp failure. For the carrier positioning mechanism, a ball in a spring loaded socket uses friction to provide locking torque; however. the ball and socket are torque limited so that the ball ran slip under kick loads (125 pounds or greater). A lead screw attached to disk spring stacks is used to provide an adjustable spring force on the socket. A locking knob is attached to the lead screw to allow for hand manipulation of the lead screw.

Mechanics of wafer bonding: Effect of clamping

NASA Astrophysics Data System (ADS)

Turner, K. T.; Thouless, M. D.; Spearing, S. M.

2004-01-01

A mechanics-based model is developed to examine the effects of clamping during wafer bonding processes. The model provides closed-form expressions that relate the initial geometry and elastic properties of the wafers to the final shape of the bonded pair and the strain energy release rate at the interface for two different clamping configurations. The results demonstrate that the curvature of bonded pairs may be controlled through the use of specific clamping arrangements during the bonding process. Furthermore, it is demonstrated that the strain energy release rate depends on the clamping configuration and that using applied loads usually leads to an undesirable increase in the strain energy release rate. The results are discussed in detail and implications for process development and bonding tool design are highlighted.

21 CFR 876.5160 - Urological clamp for males.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urological clamp for males. 876.5160 Section 876...) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5160 Urological clamp for males. (a) Identification. A urological clamp for males is a device used to close the urethra of a male to...

Interactive effects of emotional and restrained eating on responses to chocolate and affect.

PubMed

Macht, Michael; Mueller, Jochen

2007-12-01

To examine differences and interactions between emotional and restrained-eating healthy adults (56 women, 53 men) were classified into emotional or restrained eaters, and persons scoring high or low on both dimensions. Participants tasted different types of chocolate (with 30, 70, 85, or 99% cocoa content) and completed questionnaires on affect and attitudes towards chocolate. Emotional eaters reported increased craving for and increased consumption of chocolate, whereas restrained eaters experienced chocolate-related guilt. However, restrained eaters rated plain chocolate (70% and 85% cocoa) as more pleasant than other groups. Persons scoring high on both dimensions showed heightened negative affect and may be prone to disturbances of eating and affect.

Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki

N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors aremore » highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.« less

Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.

PubMed

Belle, Valérie; Rouger, Sabrina; Costanzo, Stéphanie; Liquière, Elodie; Strancar, Janez; Guigliarelli, Bruno; Fournel, André; Longhi, Sonia

2008-12-01

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

Targeted mass spectrometric analysis of N-terminally truncated isoforms generated via alternative translation initiation.

PubMed

Kobayashi, Ryuji; Patenia, Rebecca; Ashizawa, Satoshi; Vykoukal, Jody

2009-07-21

Alternative translation initiation is a mechanism whereby functionally altered proteins are produced from a single mRNA. Internal initiation of translation generates N-terminally truncated protein isoforms, but such isoforms observed in immunoblot analysis are often overlooked or dismissed as degradation products. We identified an N-terminally truncated isoform of human Dok-1 with N-terminal acetylation as seen in the wild-type. This Dok-1 isoform exhibited distinct perinuclear localization whereas the wild-type protein was distributed throughout the cytoplasm. Targeted analysis of blocked N-terminal peptides provides rapid identification of protein isoforms and could be widely applied for the general evaluation of perplexing immunoblot bands.

Tool and Technique for Restraining Live-Captured American Martens and Fishers

Treesearch

Linda Ebel Thomasma; Rolf O. Peterson; Rolf O. Peterson

1998-01-01

Restraining live-captured animals poses challenges when working alone, especially in remote field locations. While studying American martens (Marfes americana) and fishers (Martes pennantr) in Michigan, we developed a new tool, trap combs, to restrain a live-captured animal in the trap. The construction and use of trap combs are described.

Timing of clamping and factors associated with iron stores in full-term newborns

PubMed Central

Oliveira, Fabiana de Cássia Carvalho; Assis, Karine Franklin; Martins, Mariana Campos; do Prado, Mara Rúbia Maciel Cardoso; Ribeiro, Andréia Queiroz; Sant’Ana, Luciana Ferreira da Rocha; Priore, Silvia Eloiza; Franceschini, Sylvia do Carmo Castro

2014-01-01

OBJECTIVE To analyze the impact of timing of clamping and obstetric, biological and socioeconomic factors on the iron stores of full-term newborns. METHODS Cross-sectional study between October 2011 and July 2012 in which hematological parameters were evaluated for newborns in Viçosa, MG, Southeastern Brazil. It involved collecting 7 mL of umbilical cord blood from 144 full-term not underweight newborns. The parameters investigated were complete blood count, serum iron, ferritin and C-reactive protein. The time of umbilical cord clamping was measured using a digital timer without interfering in the procedures of childbirth. The birth data were collected from Live Birth Certificates and other information was obtained from the mother through a questionnaire applied in the first month postpartum. Analysis of multiple linear regression was then used to estimate the influence of biological, obstetrics and socioeconomic factors on the ferritin levels at birth. RESULTS The median ferritin was 130.3 µg/L (n = 129, minimum = 16.4; maximum = 420.5 µg/L), the mean serum iron was 137.9 μg/dL (n = 144, SD = 39.29) and mean hemoglobin was 14.7 g/dL (n = 144, SD = 1.47). The median time of cord clamping was 36 seconds, ranging between 7 and 100. The bivariate analysis detected an association between ferritin levels and color of the child, timing clamping of 60 seconds, type of delivery, the presence of gestational diabetes and per capita family income. In multivariate analysis, the variables per capita income, number of antenatal visits and length at birth accounted for 22.0% of variation in ferritin levels. CONCLUSIONS Iron stores at birth were influenced by biological, obstetric and social characteristics. Tackling anemia should involve creating policies aimed at reducing social inequalities, improving the quality of antenatal care, as well as implementing a criterion of delayed clamping of the umbilical cord within the guidelines of labor. PMID:24789632

High-disinhibition restrained eaters are disinhibited by self-regulatory depletion in the food-related inhibitory control.

PubMed

Zhou, Yizhou; Gao, Xiao; Chen, Hong; Kong, Fanchang

2017-08-01

Restrained eating for weight control and loss is becoming highly prevalent in many affluent societies, while most of the restrained eaters are rather unsuccessful in the long term. According to the strength model of self-control, the disinhibition effect of restrained eaters may occur after the depletion of self-control resources. However, no work has examined the direct impact of self-control resources on inhibitory control ability of restrained eaters. This study investigated the influences of self-control resources on the food-related inhibitory control among high-restraint/low-disinhibition restrained eaters, high-restraint/high-disinhibition restrained eaters and unrestrained eaters using stop signal task. Results reveal that there's no difference of food-related inhibitory control between three groups when the self-control resources are non-depleted, while high-restraint/high-disinhibition restrained eaters showing a decrease of food-related inhibitory control after ego-depletion. This disinhibition effect only seems to occur in samples of restrained eaters with a high tendency toward overeating. Copyright © 2017 Elsevier Ltd. All rights reserved.

Renal functional and perioperative outcomes of off-clamp versus clamped robot-assisted partial nephrectomy: matched cohort study.

PubMed

Tanagho, Youssef S; Bhayani, Sam B; Sandhu, Gurdarshan S; Vaughn, Nicholas P; Nepple, Kenneth G; Figenshau, R Sherburne

2012-10-01

To evaluate the potential benefit of performing off-clamp robot-assisted partial nephrectomy as it relates to renal functional outcomes, while assessing the safety profile of this unconventional surgical approach. Twenty-nine patients who underwent off-clamp robot-assisted partial nephrectomy for suspected renal cell carcinoma at Washington University between March 2008 and September 2011 (group 1) were matched to 29 patients with identical nephrometry scores and comparable baseline renal function who underwent robot-assisted partial nephrectomy with hilar clamping during the same period (group 2). The matched cohorts' perioperative and renal functional outcomes were compared at a mean 9-month follow-up. Mean estimated blood loss was 146.4 mL in group 1, versus 103.9 mL in group 2 (P = .039). Mean hilar clamp time was 0 minutes in group 1 and 14.7 minutes in group 2. No perioperative complications were encountered in group 1; 1 Clavien-2 complication (3.4%) occurred in group 2 (P = 1.000). At 9-month follow-up, mean estimated glomerular filtration rate in group 1 was 79.9 versus 84.8 mL/min/1.73 m(2) preoperatively (P = .013); mean estimated glomerular filtration rate in group 2 was 74.1 versus 85.8 mL/min/1.73 m(2) preoperatively (P < .001). Hence, estimated glomerular filtration rate declined by a mean of 4.9 mL/min/1.73 m(2) in group 1 versus 11.7 mL/min/1.73 m(2) in group 2 (P = .033). Off-clamp robot-assisted partial nephrectomy is associated with a favorable morbidity profile and relatively greater renal functional preservation compared to clamped robot-assisted partial nephrectomy. Nevertheless, the benefit is small in renal functional terms and may have very limited clinical relevance. Copyright © 2012 Elsevier Inc. All rights reserved.

DETAIL VIEW OF TRAM BUCKET FRAME, SHOWING CLAMPING MECHANISM,WITHOUT ORE ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

DETAIL VIEW OF TRAM BUCKET FRAME, SHOWING CLAMPING MECHANISM,WITHOUT ORE BUCKET AND WHEELS. THE FRAME IS LYING ON ITS SIDE. THE ORE BUCKET WAS ATTACHED TO THE LEFT SIDE, AND TWO WHEELS WERE ATTACHED TO THE SPINDLE ON THE RIGHT. THE FRAME AND BUCKET ARE SUSPENDED FROM THE STATIONARY CABLE BY THE TWO WHEELS, WITH THE ORE BUCKET HANGING BELOW. THE WHEEL AND LEVER AT CENTER WERE ACTIVATED BY THE OPENING AND CLOSING MECHANISMS ON THE TRAM TERMINALS TO LOCK OR RELEASE THE BUCKET ONTO THE MOVING CABLE THAT RAN THROUGH THE SQUARE BLOCK AT CENTER. - Keane Wonder Mine, Park Route 4 (Daylight Pass Cutoff), Death Valley Junction, Inyo County, CA

Young Adolescent Batterers: A Profile of Restraining Order Defendants in Massachusetts.

ERIC Educational Resources Information Center

Cochran, Donald; And Others

Research indicates a national trend of increasing violence among the juvenile population. This study examines and profiles adolescent restraining order defendants in one state. The study includes all adolescent defendants between the ages of 11 and 17 who had a restraining order issued against them during a 10-month period. This study examined the…

Na+ current in presynaptic terminals of the crayfish opener cannot initiate action potentials.

PubMed

Lin, Jen-Wei

2016-01-01

Action potential (AP) propagation in presynaptic axons of the crayfish opener neuromuscular junction (NMJ) was investigated by simultaneously recording from a terminal varicosity and a proximal branch. Although orthodromically conducting APs could be recorded in terminals with amplitudes up to 70 mV, depolarizing steps in terminals to -20 mV or higher failed to fire APs. Patch-clamp recordings did detect Na(+) current (INa) in most terminals. The INa exhibited a high threshold and fast activation rate. Local perfusion of Na(+)-free saline showed that terminal INa contributed to AP waveform by slightly accelerating the rising phase and increasing the peak amplitude. These findings suggest that terminal INa functions to "touch up" but not to generate APs. Copyright © 2016 the American Physiological Society.

Effect of sterilization on stiffness and dimensional stability of rubber-dam clamps.

PubMed

Giebink, D L; Mathieu, G P; Hondrum, S O

1996-01-01

Simulated clinical conditions were used to test the effect of sterilization on rubber-dam clamp stiffness and dimension. Sixty Hygienic and Ivory W7 clamps were either steam or dry heat sterilized and compared to controls. Stiffness and dimensional change between Ivory clamp groups was significant (p<.0001); the sterilized clamps showed less change than the controls. Hygienic groups showed a significant different between the control and dry heat groups (p<.05); the sterilized clamps showed less change than the controls. The change in stiffness and interjaw width for all Ivory clamps compared to all Hygienic clamps was significant (p<.0001). The Hygienic clamps changes less than the Ivory clamps. The results indicate that steam and dry heat sterilization do not affect retention of rubber-dam clamps.

Adverse impact of intermittent portal clamping on long-term postoperative outcomes in hepatocellular carcinoma.

PubMed

Hao, S; Chen, S; Yang, X; Wan, C

2017-01-01

Introduction To evaluate the impact of intermittent portal clamping (IPC) on long-term postoperative outcomes in patients with hepatocellular carcinoma (HCC). Methods Clinical records of 355 patients underwent curative liver resection for HCC in January 2007 to December 2010 were retrospectively reviewed. According to how portal clamping was performed, patients were grouped as: IPC, n=113; other portal clamping (OPC), n=190; and no portal clamping (NPC), n=52. Results Median recurrence-free survival (RFS) was statistically significantly shorter in the IPC (39.4 months) than OPC (47.3 months, p=0.010) and NPC groups (51.4 months, p=0.008). Median overall survival (OS) was also significantly shorter with IPC (46.3 months), versus 52.9 months with OPC (p=0.022) and 56.2 months with NPC (p=0.015). Kaplan-Meier survival analysis revealed that 5-year cumulative RFS was much lower in the IPC (42.5%) than OPC (50.9%, p=0.014) and NPC groups (49.6%, p=0.013). Five-year cumulative OS was also much lower in the IPC (44.9%) than OPC (58.0%, p=0.020) and NPC groups (57.7%, p=0.025). On univariate analysis, tumour grade, size and number, TNM stage, blood transfusion, vascular invasion and IPC were significantly inversely correlated with RFS and OS. On multivariate analysis, tumour size and number, blood transfusion, vascular invasion and IPC remained significant. Conclusions Our study suggests that IPC is an independent risk factor for poor long-term postoperative outcomes in patients with HCC.

Focusing on media body ideal images triggers food intake among restrained eaters: a test of restraint theory and the elaboration likelihood model.

PubMed

Boyce, Jessica A; Kuijer, Roeline G

2014-04-01

Although research consistently shows that images of thin women in the media (media body ideals) affect women negatively (e.g., increased weight dissatisfaction and food intake), this effect is less clear among restrained eaters. The majority of experiments demonstrate that restrained eaters - identified with the Restraint Scale - consume more food than do other participants after viewing media body ideal images; whereas a minority of experiments suggest that such images trigger restrained eaters' dietary restraint. Weight satisfaction and mood results are just as variable. One reason for these inconsistent results might be that different methods of image exposure (e.g., slideshow vs. film) afford varying levels of attention. Therefore, we manipulated attention levels and measured participants' weight satisfaction and food intake. We based our hypotheses on the elaboration likelihood model and on restraint theory. We hypothesised that advertent (i.e., processing the images via central routes of persuasion) and inadvertent (i.e., processing the images via peripheral routes of persuasion) exposure would trigger differing degrees of weight dissatisfaction and dietary disinhibition among restrained eaters (cf. restraint theory). Participants (N = 174) were assigned to one of four conditions: advertent or inadvertent exposure to media or control images. The dependent variables were measured in a supposedly unrelated study. Although restrained eaters' weight satisfaction was not significantly affected by either media exposure condition, advertent (but not inadvertent) media exposure triggered restrained eaters' eating. These results suggest that teaching restrained eaters how to pay less attention to media body ideal images might be an effective strategy in media-literary interventions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Force-controlled patch clamp of beating cardiac cells.

PubMed

Ossola, Dario; Amarouch, Mohamed-Yassine; Behr, Pascal; Vörös, János; Abriel, Hugues; Zambelli, Tomaso

2015-03-11

From its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening because it is the only tool enabling accurate investigation of voltage-gated ion channels, which are responsible for action potentials. Because of its key role in drug screening, innovation efforts are being made to reduce its complexity toward more automated systems. While some of these new approaches are being adopted in pharmaceutical companies, conventional patch-clamp remains unmatched in fundamental research due to its versatility. Here, we merged the patch clamp and atomic force microscope (AFM) techniques, thus equipping the patch-clamp with the sensitive AFM force control. This was possible using the FluidFM, a force-controlled nanopipette based on microchanneled AFM cantilevers. First, the compatibility of the system with patch-clamp electronics and its ability to record the activity of voltage-gated ion channels in whole-cell configuration was demonstrated with sodium (NaV1.5) channels. Second, we showed the feasibility of simultaneous recording of membrane current and force development during contraction of isolated cardiomyocytes. Force feedback allowed for a gentle and stable contact between AFM tip and cell membrane enabling serial patch clamping and injection without apparent cell damage.

Stress-induced release of GUT peptides in young women classified as restrained or unrestrained eaters.

PubMed

Hilterscheid, Esther; Laessle, Reinhold

2015-12-01

Basal release of GUT peptides has been found to be altered in restrained eaters. Stress-induced secretion, however, has not yet been described, but could be a biological basis of overeating that exposes restrained eaters to a higher risk of becoming obese. The aim of the present study was to compare restrained and unrestrained eaters with respect to stress-induced release of the GUT peptides ghrelin and PYY. 46 young women were studied. Blood sampling for peptides was done before and after the Trier Social Stress Test. Ghrelin secretion after stress was significantly elevated in the restrained eaters, whereas no significant differences were detected for PYY. Stress-induced release of GUT peptides can be interpreted as a cause as well as a consequence of restrained eating.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

The influence of air bags and restraining devices on extremity injuries in motor vehicle collisions.

PubMed

McGovern, M K; Murphy, R X; Okunski, W J; Wasser, T E

2000-05-01

The influence of air bags and other restraining devices on injury after motor vehicle collisions is not well defined. This study examined the relationship between the use of restraining devices and the incidence of extremity injuries in motor vehicle collisions. A retrospective analysis was performed on motor vehicle collision data submitted to the Pennsylvania Trauma Outcome Study database from 1990 through 1995. Criteria for submission included trauma patients who were admitted to the intensive care unit, who died during hospitalization, who were hospitalized for more than 72 hours, or who were transferred in or out of the receiving hospital. A total of 21,875 patients met these criteria. These patients were analyzed for the presence or absence of upper and lower extremity injuries and were compared based on their use of restraining devices. Restraining devices were categorized into four groups: air bag alone, air bag and seat belt, seat belt or carseat without air bag, and no restraining device. Statistical analysis was performed using the chi-squared test of association. For contingency tables with small expected frequencies, Fisher's exact test was used. Study participants included 11,688 men and 10,185 women with a mean age of 38 +/- 20 years. There were 16,033 drivers and 5,842 passengers. Air bags were deployed in 472 instances. In 297 of these cases, additional restraint was provided with a seat belt. In 6,632 cases, air bags were not deployed; however, patients were restrained with either a seat belt or a carseat. In 14,771 cases, patients were not restrained. When comparing restraining devices as a group vs. no restraint, there was a significant decrease in the incidence of upper (p = 0.018) and lower (p < 0.001) extremity injuries. Air bags, however, were associated with an increased incidence of both upper (p = 0.033) and lower (p = 0.002) extremity injuries when compared with no restraint or when compared among patients who were restrained. As a group

The C- and N-Terminal Residues of Synthetic Heptapeptide Ion Channels Influence Transport Efficacy Through Phospholipid Bilayers

PubMed Central

Djedovič, Natasha; Ferdani, Riccardo; Harder, Egan; Pajewska, Jolanta; Pajewski, Robert; Weber, Michelle E.; Schlesinger, Paul H.; Gokel, George W.

2008-01-01

The synthetic peptide, R2N-COCH2OCH2CO-Gly-Gly-Gly-Pro-Gly-Gly-Gly-OR’, was shown to be selective for Cl- over K+ when R is n-octadecyl and R’ is benzyl. Nineteen heptapeptides have now been prepared in which the N-terminal and C-terminal residues have been varied. All of the N-terminal residues are dialkyl but the C-terminal chains are esters, 2° amides, or 3° amides. The compounds having varied N-terminal anchors and C-terminal benzyl groups are as follows: 1, R = n-propyl; 2, R = n-hexyl; 3, R = n-octyl; 4, R = n-decyl; 5, R = n-dodecyl; 6, R = n-tetradecyl; 7, R = n-hexadecyl; 8, R = n-octadecyl. Compounds 9-19 have R = n-octadecyl and C-terminal residues as follows: 9, OR’ = OCH2CH3; 10, OR’ = OCH(CH3)2; 11, OR’ = O(CH2)6CH3; 12, OR’ = OCH2-c-C6H11; 13, OR’ = O(CH2)9CH3; 14, OR’ = O (CH2)17CH3; 15, NR’2 = N[(CH2)6CH3]2; 16, NHR’ = NH(CH2)9CH3; 17, NR’2 = N[(CH2)9CH3]2; 18, NHR’ = NH(CH2)17CH3; 19, NR’2 = N[(CH2)17CH3]2. The highest anion transport activities were observed as follows. For the benzyl esters whose N-terminal residues were varied, i.e. 1-8, compound 3 was most active. For the C18 anchored esters 10-14, n-heptyl ester 11 was most active. For the C18 anchored, C-terminal amides 15-19, di-n-decylamide 17 was most active. It was concluded that both the C- and N-terminal anchors were important for channel function in the bilayer but that activity was lost unless only one of the two anchoring groups was dominant. PMID:19633728

Resveratrol Treatment after Status Epilepticus Restrains Neurodegeneration and Abnormal Neurogenesis with Suppression of Oxidative Stress and Inflammation.

PubMed

Mishra, Vikas; Shuai, Bing; Kodali, Maheedhar; Shetty, Geetha A; Hattiangady, Bharathi; Rao, Xiaolan; Shetty, Ashok K

2015-12-07

Antiepileptic drug therapy, though beneficial for restraining seizures, cannot thwart status epilepticus (SE) induced neurodegeneration or down-stream detrimental changes. We investigated the efficacy of resveratrol (RESV) for preventing SE-induced neurodegeneration, abnormal neurogenesis, oxidative stress and inflammation in the hippocampus. We induced SE in young rats and treated with either vehicle or RESV, commencing an hour after SE induction and continuing every hour for three-hours on SE day and twice daily thereafter for 3 days. Seizures were terminated in both groups two-hours after SE with a diazepam injection. In contrast to the vehicle-treated group, the hippocampus of animals receiving RESV during and after SE presented no loss of glutamatergic neurons in hippocampal cell layers, diminished loss of inhibitory interneurons expressing parvalbumin, somatostatin and neuropeptide Y in the dentate gyrus, reduced aberrant neurogenesis with preservation of reelin + interneurons, lowered concentration of oxidative stress byproduct malondialdehyde and pro-inflammatory cytokine tumor necrosis factor-alpha, normalized expression of oxidative stress responsive genes and diminished numbers of activated microglia. Thus, 4 days of RESV treatment after SE is efficacious for thwarting glutamatergic neuron degeneration, alleviating interneuron loss and abnormal neurogenesis, and suppressing oxidative stress and inflammation. These results have implications for restraining SE-induced chronic temporal lobe epilepsy.

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, G.; Kirouac, K.; Shin, Y.J.

2009-09-16

DNA polymerases are co-ordinated by sliding clamps (PCNA/{beta}-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 {angstrom} resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with amore » 3{sub 10} helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.« less

Design and experimental research of a novel inchworm type piezo-driven rotary actuator with the changeable clamping radius.

PubMed

Zhao, Hongwei; Fu, Lu; Ren, Luquan; Huang, Hu; Fan, Zunqiang; Li, Jianping; Qu, Han

2013-01-01

In this paper, a novel piezo-driven rotary actuator with the changeable clamping radius is developed based on the inchworm principle. This actuator mainly utilizes three piezoelectric actuators, a flexible gripper, a clamping block, and a rotor to achieve large stroke rotation with high resolution. The design process of the flexible gripper consisting of the driving unit and the clamping unit is described. Lever-type mechanisms were used to amplify the micro clamping displacements. The amplifying factor and parasitic displacement of the lever-type mechanism in the clamping unit was analyzed theoretically and experimentally. In order to investigate the rotation characteristics of the actuator, a series of experiments was carried out. Experimental results indicate that the actuator can rotate at a speed of 77,488 μrad/s with a driving frequency of 167 Hz. The rotation resolution and maximum load torque of the actuator are 0.25 μrad and 37 N mm, respectively. The gripper is movable along the z direction based on an elevating platform, and the clamping radius can change from 10.6 mm to 25 mm. Experimental results confirm that the actuator can achieve different rotation speeds by changing the clamping radius.

Involvement of the N-terminal region in alpha-crystallin-lens membrane recognition

NASA Technical Reports Server (NTRS)

Ifeanyi, F.; Takemoto, L.; Spooner, B. S. (Principal Investigator)

1991-01-01

Previous studies have demonstrated that alpha-crystallin binds specifically, in a saturable manner, to lens membrane. To determine the region of the alpha-crystallin molecule that might be involved in this binding, native alpha-crystallin from the bovine lens has been treated by limited digestion with trypsin, to produce alpha-A molecules with an intact C-terminal region, and a nicked N-terminal region. Compared to intact alpha-crystallin, trypsin-treated alpha-crystallin binds less avidly to lens membrane, suggesting that the N-terminal region of the alpha-A molecule may play a key role in the recognition between lens membrane and crystallin.

In-Operando Spatial Imaging of Edge Termination Electric Fields in GaN Vertical p-n Junction Diodes

DOE PAGES

Leonard, Francois; Dickerson, J. R.; King, M. P.; ...

2016-05-03

Control of electric fields with edge terminations is critical to maximize the performance of high-power electronic devices. We proposed a variety of edge termination designs which makes the optimization of such designs challenging due to many parameters that impact their effectiveness. And while modeling has recently allowed new insight into the detailed workings of edge terminations, the experimental verification of the design effectiveness is usually done through indirect means, such as the impact on breakdown voltages. In this letter, we use scanning photocurrent microscopy to spatially map the electric fields in vertical GaN p-n junction diodes in operando. We alsomore » reveal the complex behavior of seemingly simple edge termination designs, and show how the device breakdown voltage correlates with the electric field behavior. Modeling suggests that an incomplete compensation of the p-type layer in the edge termination creates a bilayer structure that leads to these effects, with variations that significantly impact the breakdown voltage.« less

Releasing N-glycan from peptide N-terminus by N-terminal succinylation assisted enzymatic deglycosylation.

PubMed

Weng, Yejing; Sui, Zhigang; Jiang, Hao; Shan, Yichu; Chen, Lingfan; Zhang, Shen; Zhang, Lihua; Zhang, Yukui

2015-04-22

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Lotus birth, a holistic approach on physiological cord clamping.

PubMed

Zinsser, Laura A

2018-04-01

The positive effects of delayed cord clamping (DCC) has been extensively researched. DCC means: waiting at least one minute after birth before clamping and cutting the cord or till the pulsation has stopped. With physiological clamping and cutting (PCC) the clamping and cutting can happen at the earliest after the pulsation has stopped. With a Lotus birth, no clamping and cutting of the cord is done. A woman called Clair Lotus Day imitated the holistic approach of PCC from an anthropoid ape in 1974. The chimpanzee did not separate the placenta from the newborn. The aim of this case report is to discuss and learn a different approach in the third stage of labour. Three cases of Lotus birth by human beings were observed. All three women gave birth in an out-of-hospital setting and had ambulant postnatal care. The placenta was washed, salted and herbs were put on 2-3h post partum. The placenta was wrapped in something that absorbs the moisture. The salting was repeated with a degreasing frequency depending on moistness of the placenta. On life day six all three Lotus babies experiences a natural separation of the cord. All three Lotus birth cases were unproblematic, no special incidence occurred. One should differentiate between early cord clamping (ECC), delayed cord clamping (DCC) and physiological cord clamping (PCC). Lotus birth might lead to an optimisation of the bonding and attachment. Research is needed in the areas of both PCC and Lotus birth. Copyright © 2017 Australian College of Midwives. Published by Elsevier Ltd. All rights reserved.

Surface characterization of selected LDEF tray clamps

NASA Technical Reports Server (NTRS)

Cromer, T. F.; Grammer, H. L.; Wightman, J. P.; Young, Philip R.; Slemp, Wayne S.

1993-01-01

The surface characterization of chromic acid anodized 6061-T6 aluminum alloy tray clamps has shown differences in surface chemistry depending upon the position on the Long Duration Exposure Facility (LDEF). Water contact angle results showed no changes in wettability of the tray clamps. The overall surface topography of the control, trailing edge(E3) and leading edge(D9) samples was similar. The thickness of the aluminum oxide layer for all samples determined by Auger depth profiling was less than one micron. X-ray photoelectron spectroscopy (XPS) analysis of the tray clamps showed significant differences in the surface composition. Carbon and silicon containing compounds were the primary contaminants detected.

π-Clamp-mediated cysteine conjugation

NASA Astrophysics Data System (ADS)

Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

2016-02-01

Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

Neurofeedback reduces overeating episodes in female restrained eaters: a randomized controlled pilot-study.

PubMed

Schmidt, Jennifer; Martin, Alexandra

2015-12-01

Overeating episodes, despite of intentions to control weight, are a common problem among women. Recurring episodes of overeating and dietary failure have been reported to result in higher Body Mass Indexes and to induce severe distress even in non-clinical groups. Based on findings from physiological research on eating behavior and craving, as well as previous biofeedback studies, we derived a cue exposure based EEG neurofeedback protocol to target overeating episodes. The treatment was evaluated in a randomized controlled trial, comparing a neurofeedback group (NFG; n = 14) with a waiting list control group (WLG; n = 13) in a sub-clinical sample of female restrained eaters. At post-treatment, the number of weekly overeating episodes and subsequent distress were significantly reduced in the NFG compared to the WLG (p < .01; r > .50). In a 3 month follow-up, effects in the NFG remained stable. As secondary outcomes, perceived dieting success was enhanced after the treatment. At follow-up, additional beneficial effects on trait food craving were observed. Altogether, we found preliminary evidence for the cue exposure neurofeedback against overeating episodes in female restrained eaters, although specific effects and underlying mechanisms still have to be explored in future research.

N-terminal pro-brain natriuretic peptide in acute Kawasaki disease correlates with coronary artery involvement.

PubMed

Adjagba, Philippe M; Desjardins, Laurent; Fournier, Anne; Spigelblatt, Linda; Montigny, Martine; Dahdah, Nagib

2015-10-01

We have lately documented the importance of N-terminal pro-brain natriuretic peptide in aiding the diagnosis of Kawasaki disease. We sought to investigate the potential value of N-terminal pro-brain natriuretic peptide pertaining to the prediction of coronary artery dilatation (Z-score>2.5) and/or of resistance to intravenous immunoglobulin therapy. We hypothesised that increased serum N-terminal pro-brain natriuretic peptide level correlates with increased coronary artery dilatation and/or resistance to intravenous immunoglobulin. We carried out a prospective study involving newly diagnosed patients treated with 2 g/kg intravenous immunoglobulin within 5-10 days of onset of fever. Echocardiography was performed in all patients at onset, then weekly for 3 weeks, then at month 2, and month 3. Coronary arteries were measured at each visit, and coronary artery Z-score was calculated. All the patients had N-terminal pro-brain natriuretic peptide serum level measured at onset, and the Z-score calculated. There were 109 patients enrolled at 6.58±2.82 days of fever, age 3.79±2.92 years. High N-terminal pro-brain natriuretic peptide level was associated with coronary artery dilatation at onset in 22.2 versus 5.6% for normal N-terminal pro-brain natriuretic peptide levels (odds ratio 4.8 [95% confidence interval 1.05-22.4]; p=0.031). This was predictive of cumulative coronary artery dilatation for the first 3 months (p=0.04-0.02), but not during convalescence at 2-3 months (odds ratio 1.28 [95% confidence interval 0.23-7.3]; p=non-significant). Elevated N-terminal pro-brain natriuretic peptide levels did not predict intravenous immunoglobulin resistance, 15.3 versus 13.5% (p=1). Elevated N-terminal pro-brain natriuretic peptide level correlates with acute coronary artery dilatation in treated Kawasaki disease, but not with intravenous immunoglobulin resistance.

Structural and kinematic evolution of a Miocene to Recent sinistral restraining bend: the Montejunto massif, Portugal

NASA Astrophysics Data System (ADS)

Curtis, Michael L.

1999-01-01

The Montejunto massif lies in the apex of a large-scale restraining bend at the southern termination of a sinistral transpressive fault system, in the Lusitanian basin of Portugal. Cenozoic deformation within the Montejunto massif initiated with southerly directed thrusting along the southern boundary of the massif, in association with the development of the E-W oriented Montejunto anticline, probably during the Langhian. Deformation switched to the northern boundary of the massif, in association with a change to NW-directed thrusting and continued development of the Montejunto anticline. The youngest set of structures within the massif is related to the sinistral reactivation of the Arieiro fault system, and steeply inclined bedding. This late phase of deformation represents the accommodation of a component of sinistral displacement across the restraining bend along mechanical anisotropies formed during this progressive Cenozoic deformation event. Variation in the kinematic style of the Main Arieiro fault is related to the angle ( α) between the fault plane and the displacement vector. Where α≈20°, abrupt pene-contemporaneous switches in displacement direction are recorded along the fault, whereas strike-slip kinematics predominate where α<20°. The timing of deformation events in the Montejunto massif is uncertain. However, correlation with the established Cenozoic Africa/Europe plate convergence directions may provide potential temporal constraints.

RESTRAINING SYSTEM FOR PLETHYSMOGRAPHY IN SMALL ANIMALS

EPA Science Inventory

A restraining technique for immobilizing awake guinea pigs and rats during physiological measurements is described. The basic device consists of an adjustable acrylic holder that supports the animal in a standing position while restricting movement of the four legs. The holder al...

Magnetic field response of doubly clamped magnetoelectric microelectromechanical AlN-FeCo resonators

NASA Astrophysics Data System (ADS)

Bennett, S. P.; Baldwin, J. W.; Staruch, M.; Matis, B. R.; LaComb, J.; van't Erve, O. M. J.; Bussmann, K.; Metzler, M.; Gottron, N.; Zappone, W.; LaComb, R.; Finkel, P.

2017-12-01

Magnetoelectric (ME) cantilever resonators have been successfully employed as magnetic sensors to measure low magnetic fields; however, high relative resolution enabling magnetometry in high magnetic fields is lacking. Here, we present on-chip silicon based ME microelectromechanical (MEMS) doubly clamped resonators which can be utilized as high sensitivity, low power magnetic sensors. The resonator is a fully suspended thin film ME heterostructure composed of an active magnetoelastic layer (Fe0.3Co0.7), which is strain coupled to a piezoelectric signal/excitation layer (AlN). By controlling uniaxial stress arising from the large magnetoelastic properties of magnetostrictive FeCo, a magnetically driven shift of the resonance frequency of the first fundamental flexural mode is observed. The theoretical intrinsic magnetic noise floor of such sensors reaches a minimum value of 35 p T /√{H z }. This approach shows a magnetic field sensitivity of ˜5 Hz/mT in a bias magnetic field of up to 120 mT. Such sensors have the potential in applications required for enhanced dynamic sensitivity in high-field magnetometry.

Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

NASA Technical Reports Server (NTRS)

Cromwell, B. K.; Shepherd, S. D.; Pender, C. W.; Wood, B. E.

1993-01-01

Infrared hemispherical reflectance measurements that were made on 58 chromic acid anodized tray clamps from LDEF are described. The measurements were made using a hemiellipsoidal mirror reflectometer with interferometer for wavelengths between 2-15 microns. The tray clamps investigated were from locations about the entire spacecraft and provided the opportunity for comparing the effects of atomic oxygen at each location. Results indicate there was essentially no dependence on atomic oxygen fluence for the surfaces studied, but there did appear to be a slight dependence on solar radiation exposure. The reflectances of the front sides of the tray clamps consistently were slightly higher than for the protected rear tray clamp surfaces.

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2014-10-01

AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH

Evaluation of Introduction of a Delayed Cord Clamping Protocol for Premature Neonates in a High-Volume Maternity Center.

PubMed

Liu, Lilly Y; Feinglass, Joe M; Khan, Janine Y; Gerber, Susan E; Grobman, William A; Yee, Lynn M

2017-05-01

To evaluate adherence to a delayed cord clamping protocol for preterm births in the first 2 years after its introduction, perform a quality improvement assessment, and determine neonatal outcomes associated with protocol implementation and adherence. This is a retrospective cohort study of women delivering singleton neonates at 23-32 weeks of gestation in the 2 years before (preprotocol) and 2 years after (postprotocol) introduction of a 30-second delayed cord clamping protocol at a large-volume academic center. This policy was communicated to obstetric and pediatric health care providers and nurses and reinforced with intermittent educational reviews. Barriers to receiving delayed cord clamping were assessed using χ tests and multivariable logistic regression. Neonatal outcomes then were compared between all neonates in the preprotocol period and all neonates in the postprotocol period and between all neonates in the preprotocol period and neonates receiving delayed cord clamping in the postprotocol period using multivariable linear and logistic regression analyses. Of the 427 eligible neonates, 187 were born postprotocol. Of these, 53.5% (n=100) neonates received delayed cord clamping according to the protocol. The rate of delayed cord clamping preprotocol was 0%. Protocol uptake and frequency of delayed cord clamping increased over the 2 years after its introduction. In the postprotocol period, cesarean delivery was the only factor independently associated with failing to receive delayed cord clamping (adjusted odds ratio [OR] 0.49, 95% confidence interval [CI] 0.25-0.96). In comparison with the preprotocol period, those who received delayed cord clamping in the postprotocol period had significantly higher birth hematocrit (β=2.46, P=.007) and fewer blood transfusions in the first week of life (adjusted OR 0.49, 95% CI 0.25-0.96). After introduction of an institutional delayed cord clamping protocol followed by continued health care provider education and

Planar patch clamp for neuronal networks--considerations and future perspectives.

PubMed

Bosca, Alessandro; Martina, Marzia; Py, Christophe

2014-01-01

The patch-clamp technique is generally accepted as the gold standard for studying ion channel activity allowing investigators to either "clamp" membrane voltage and directly measure transmembrane currents through ion channels, or to passively monitor spontaneously occurring intracellular voltage oscillations. However, this resulting high information content comes at a price. The technique is labor-intensive and requires highly trained personnel and expensive equipment. This seriously limits its application as an interrogation tool for drug development. Patch-clamp chips have been developed in the last decade to overcome the tedious manipulations associated with the use of glass pipettes in conventional patch-clamp experiments. In this chapter, we describe some of the main materials and fabrication protocols that have been developed to date for the production of patch-clamp chips. We also present the concept of a patch-clamp chip array providing high resolution patch-clamp recordings from individual cells at multiple sites in a network of communicating neurons. On this chip, the neurons are aligned with the aperture-probes using chemical patterning. In the discussion we review the potential use of this technology for pharmaceutical assays, neuronal physiology and synaptic plasticity studies.

Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

PubMed Central

Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

2017-01-01

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

30 CFR 75.605 - Clamping of trailing cables to equipment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Clamping of trailing cables to equipment. 75... MINE SAFETY AND HEALTH MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trailing Cables § 75.605 Clamping of trailing cables to equipment. [Statutory Provisions] Trailing cables shall be clamped to...

Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells

PubMed Central

Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

2017-01-01

Heat shock protein 90 (Hsp90) contains amino (N)–terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states. PMID:28036294

Effects of funnel web spider toxin on Ca2+ currents in neurohypophysial terminals.

PubMed

Wang, G; Lemos, J R

1994-11-14

Funnel web spider toxin (FTX) is reportedly a specific blocker of P-type Ca2+ channels. The effects of FTX on the Ca2+ currents of isolated neurohypophysial nerve terminals of the rat were investigated using the 'whole-cell' patch-clamp technique. Both the transient and long-lasting Ca2+ current components were maximally elicited by depolarization from a holding potential equal to the normal terminal resting potential (-90 mV). Externally applied FTX inhibited the high-voltage-threshold, transient component of the Ca2+ current in a concentration-dependent manner, with a half-maximal inhibition at a dilution of approximately 1:10000. FTX also shifted the peak current of the I-V relationship by +10 mV. The long-lasting Ca2+ current component, which is sensitive to L-type Ca2+ channel blockers, was insensitive to FTX. The transient current, which is sensitive to omega-conotoxin GVIA, was completely blocked by FTX. These results suggest that there could be a novel, inactivating Ca2+ channel in the rat neurohypophysial terminals which is affected by both N-type and P-type Ca2+ channel blockers.

An Ultrasonic Clamp for Bloodless Partial Nephrectomy

NASA Astrophysics Data System (ADS)

Lafon, Cyril; Bouchoux, Guillaume; Murat, François Joseph; Birer, Alain; Theillère, Yves; Chapelon, Jean Yves; Cathignol, Dominique

2007-05-01

Maximum conservation of the kidney is preferable through partial nephrectomy for patients at risk of disease recurrence of renal cancers. Haemostatic tools are needed in order to achieve bloodless surgery and reduce post surgery morbidity. Two piezo-ceramic transducers operating at a frequency of 4 MHz were mounted on each arm of a clamp. When used for coagulation purposes, two transducers situated on opposite arms of the clamp were driven simultaneously. Heat delivery was optimized as each transducers mirrored back to targeted tissues the wave generated by the opposite transducer. Real-time treatment monitoring with an echo-based technique was also envisaged with this clamp. Therapy was periodically interrupted so one transducer could generate a pulse. The echo returning from the opposite transducer was treated. Coagulation necroses were obtained in vitro on substantial thicknesses (23-38mm) of pig liver over exposure durations ranging from 30s to 130s, and with acoustic intensities of less than 15W/cm2 per transducer. Both kidneys of two pigs were treated in vivo with the clamp (14.5W/cm2 for 90s), and the partial nephrectomies performed proved to be bloodless. In vitro and in vivo, wide transfixing lesions corresponded to an echo energy decrease superior to -10dB and parabolic form of the time of flight versus treatment time. In conclusion, this ultrasound clamp has proven to be an excellent mean for achieving monitored haemostasis in kidney.

The diagnostic value of plasma N-terminal connective tissue growth factor levels in children with heart failure.

PubMed

Li, Gang; Song, Xueqing; Xia, Jiyi; Li, Jing; Jia, Peng; Chen, Pengyuan; Zhao, Jian; Liu, Bin

2017-01-01

The aim of this study was to assess the diagnostic value of plasma N-terminal connective tissue growth factor in children with heart failure. Methods and results Plasma N-terminal connective tissue growth factor was determined in 61 children, including 41 children with heart failure, 20 children without heart failure, and 30 healthy volunteers. The correlations between plasma N-terminal connective tissue growth factor levels and clinical parameters were investigated. Moreover, the diagnostic value of N-terminal connective tissue growth factor levels was evaluated. Compared with healthy volunteers and children without heart failure, plasma N-terminal connective tissue growth factor levels were significantly elevated in those with heart failure (p0.05), but it obviously improved the ability of diagnosing heart failure in children, as demonstrated by the integrated discrimination improvement (6.2%, p=0.013) and net re-classification improvement (13.2%, p=0.017) indices. Plasma N-terminal connective tissue growth factor is a promising diagnostic biomarker for heart failure in children.

Head impact contact points for restrained child occupants.

PubMed

Arbogast, Kristy B; Wozniak, Samantha; Locey, Caitlin M; Maltese, Matthew R; Zonfrillo, Mark R

2012-01-01

Head injuries are the most common injuries sustained by children in motor vehicle crashes regardless of age, restraint, and crash direction. For rear seat occupants, the interaction of the subject with the seat back and the vehicle side interior structures has been previously highlighted. In order to advance this knowledge to the development of countermeasures, a summary of vehicle components that contributed to these injuries is needed. Therefore, the objective of this study was to create a contact map of the vehicle interior for head and face injuries to rear-seated restrained children in front crashes. The Crash Injury Research and Engineering Network (CIREN) was queried for rear-seated, restrained child occupants (age 0-15 years) in forward-facing child restraints, booster seats, or lap and shoulder belts who sustained an AIS2+ head and/or face injury in a frontal motor vehicle crash. Cases were analyzed to describe injury patterns and injury causation scenarios. A contact point map was developed to summarize the vehicle components related to injury causation of the head/face injury. Twenty-one cases met the combined inclusion and exclusion criteria. Seven of the child occupants were restrained in forward-facing child restraints, 2 in belt-positioning booster seats, and 12 in lap and shoulder belts. There were 28 head and 17 facial injuries. For left rear occupants, the most common contact point was the pillar in front of the occupant's seat row; that is, B-pillar for second-row occupants, indicating a leftward kinematics. For right rear occupants, due to differences in crash dynamics, the most common contact point location was the passenger's seat back, suggesting that these occupants moved predominantly forward. Contact points associated with head/face injury for restrained children 0 to 15 years in frontal crashes have been delineated. In a majority of the cases, the head/face injury was the most severe injury and severe injuries to other body regions were

Ulcers in restrained rats: Study of protective substances

NASA Technical Reports Server (NTRS)

Buche, L.; Gallaire, D.

1980-01-01

The genesis of ulcers in restrained rats is discussed through an investigation of the relationship between the protective effects of nervous system effectual substances examined vis-a-vis ulcers in restrained rats and their elective or secondary pharmacologic effects. The substances used were capable of either peripheral parasympatholytic, sympatholytic, ganglioplegic, spasmolytic effects or central, hypnotic, tranquilizing, neuroleptic, analgesic effects. The regular and considerable protection observed with parasympatholytics (atropine sulfate, benzylonium bromide, dihexyverine, J.L. 1344) and a ganglioplegic (pentamethonium) is a function of their anticholinergic properties. It is of less importance with dibenamine, a sympatholytic action on the adrenergic receptors. Among the central depressive substances tested (hypnotics, tranquilizers, neuroleptics, analgesic), phenobarbital at a nonhypnotic dose, and dextromoramide at a nonanalgesic dose, show antiulcerous effects, which are found with chlorpromazine only at cataleptogenic doses.

Research on the honeycomb restrain layer application to the high power microwave dielectric window

NASA Astrophysics Data System (ADS)

Zhang, Qingyuan; Shao, Hao; Huang, Wenhua; Guo, Letian

2018-01-01

Dielectric window breakdown is an important problem of high power microwave radiation. A honeycomb layer can suppress the multipactor in two directions to restrain dielectric window breakdown. This paper studies the effect of the honeycomb restrain layer on improving the dielectric window power capability. It also studies the multipactor suppression mechanism by using the electromagnetic particle-in-cell software, gives the design method, and accomplishes the test experiment. The experimental results indicated that the honeycomb restrain layer can effectively improve the power capability twice.

Research on the honeycomb restrain layer application to the high power microwave dielectric window.

PubMed

Zhang, Qingyuan; Shao, Hao; Huang, Wenhua; Guo, Letian

2018-01-01

Dielectric window breakdown is an important problem of high power microwave radiation. A honeycomb layer can suppress the multipactor in two directions to restrain dielectric window breakdown. This paper studies the effect of the honeycomb restrain layer on improving the dielectric window power capability. It also studies the multipactor suppression mechanism by using the electromagnetic particle-in-cell software, gives the design method, and accomplishes the test experiment. The experimental results indicated that the honeycomb restrain layer can effectively improve the power capability twice.

Carbon nanotube-clamped metal atomic chain

PubMed Central

Tang, Dai-Ming; Yin, Li-Chang; Li, Feng; Liu, Chang; Yu, Wan-Jing; Hou, Peng-Xiang; Wu, Bo; Lee, Young-Hee; Ma, Xiu-Liang; Cheng, Hui-Ming

2010-01-01

Metal atomic chain (MAC) is an ultimate one-dimensional structure with unique physical properties, such as quantized conductance, colossal magnetic anisotropy, and quantized magnetoresistance. Therefore, MACs show great potential as possible components of nanoscale electronic and spintronic devices. However, MACs are usually suspended between two macroscale metallic electrodes; hence obvious technical barriers exist in the interconnection and integration of MACs. Here we report a carbon nanotube (CNT)-clamped MAC, where CNTs play the roles of both nanoconnector and electrodes. This nanostructure is prepared by in situ machining a metal-filled CNT, including peeling off carbon shells by spatially and elementally selective electron beam irradiation and further elongating the exposed metal nanorod. The microstructure and formation process of this CNT-clamped MAC are explored by both transmission electron microscopy observations and theoretical simulations. First-principles calculations indicate that strong covalent bonds are formed between the CNT and MAC. The electrical transport property of the CNT-clamped MAC was experimentally measured, and quantized conductance was observed. PMID:20427743

Spectral infrared hemispherical reflectance measurements for LDEF tray clamps

NASA Technical Reports Server (NTRS)

Wood, Bobby E.; Cromwell, Brian K.; Pender, Charles W.; Shepherd, Seth D.

1992-01-01

This paper describes infrared hemispherical reflectance measurements (2-15 microns) that were made on 58 chromic acid anodized tray clamps retrieved from the LDEF spacecraft. These clamps were used for maintaining the experiments in place and were located at various locations about the spacecraft. Changes in reflectance of the tray clamps at these locations were compared with atomic oxygen fluxes at the same locations. A decrease in absorption band depth was seen for the surfaces exposed to space indicating that there was some surface layer erosion. In all of the surfaces measured, little evidence of contamination was observed and none of the samples showed evidence of the brown nicotine stain that was so prominent in other experiments. Total emissivity values were calculated for both exposed and unexposed tray clamp surfaces. Only small differences, usually less than 1 percent, were observed. The spectral reflectances were measured using a hemi-ellipsoidal mirror reflectometer matched with an interferometer spectrometer. The rapid scanning capability of the interferometer allowed the reflectance measurements to be made in a timely fashion. The ellipsoidal mirror has its two foci separated by 2 inches and located on the major axis. A blackbody source was located at one focus while the tray clamp samples were located at the conjugate focus. The blackbody radiation was modulated and then focused by the ellipsoid onto the tray clamps. Radiation reflected from the tray clamp was sampled by the interferometer by viewing through a hole in the ellipsoid. A gold mirror (reflectance approximately 98 percent) was used as the reference surface.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

PubMed

Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

2014-10-22

Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

Tensil Film Clamps And Mounting Block For Viscoelastometers

NASA Technical Reports Server (NTRS)

Stoakley, Diane M.; St. Clair, Anne K.; Little, Bruce D.

1989-01-01

Set of clamps and mounting block developed for use in determining tensile moduli and damping properties of films in manually operated or automated commercial viscoelastometer. These clamps and block provide uniformity of sample gripping and alignment in instrument. Dependence on operator and variability of data greatly reduced.

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

A monogenean without clamps

USDA-ARS?s Scientific Manuscript database

Ectoparasites face a daily challenge: to remain attached to their host. Polyopisthocotylean monogeneans attach to the surface of fish gills by highly specialized structures, the sclerotized clamps. In the original description of the protomicrocotylid species Lethacotyle fijiensis, described 50 years...

Dynamic analysis of clamp band joint system subjected to axial vibration

NASA Astrophysics Data System (ADS)

Qin, Z. Y.; Yan, S. Z.; Chu, F. L.

2010-10-01

Clamp band joints are commonly used for connecting circular components together in industry. Some of the systems jointed by clamp band are subjected to dynamic load. However, very little research on the dynamic characteristics for this kind of joint can be found in the literature. In this paper, a dynamic model for clamp band joint system is developed. Contact and frictional slip between the components are accommodated in this model. Nonlinear finite element analysis is conducted to identify the model parameters. Then static experiments are carried out on a scaled model of the clamp band joint to validate the joint model. Finally, the model is adopted to study the dynamic characteristics of the clamp band joint system subjected to axial harmonic excitation and the effects of the wedge angle of the clamp band joint and the preload on the response. The model proposed in this paper can represent the nonlinearity of the clamp band joint and be used conveniently to investigate the effects of the structural and loading parameters on the dynamic characteristics of this type of joint system.

Planar patch clamp: advances in electrophysiology.

PubMed

Brüggemann, Andrea; Farre, Cecilia; Haarmann, Claudia; Haythornthwaite, Ali; Kreir, Mohamed; Stoelzle, Sonja; George, Michael; Fertig, Niels

2008-01-01

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Design, Synthesis, and Evaluation of N- and C-Terminal Protein Bioconjugates as G Protein-Coupled Receptor Agonists.

PubMed

Healey, Robert D; Wojciechowski, Jonathan P; Monserrat-Martinez, Ana; Tan, Susan L; Marquis, Christopher P; Sierecki, Emma; Gambin, Yann; Finch, Angela M; Thordarson, Pall

2018-02-21

A G protein-coupled receptor (GPCR) agonist protein, thaumatin, was site-specifically conjugated at the N- or C-terminus with a fluorophore for visualization of GPCR:agonist interactions. The N-terminus was specifically conjugated using a synthetic 2-pyridinecarboxyaldehyde reagent. The interaction profiles observed for N- and C-terminal conjugates were varied; N-terminal conjugates interacted very weakly with the GPCR of interest, whereas C-terminal conjugates bound to the receptor. These chemical biology tools allow interactions of therapeutic proteins:GPCR to be monitored and visualized. The methodology used for site-specific bioconjugation represents an advance in application of 2-pyridinecarboxyaldehydes for N-terminal specific bioconjugations.

A novel high performance ESD power clamp circuit with a small area

NASA Astrophysics Data System (ADS)

Zhaonian, Yang; Hongxia, Liu; Li, Li; Qingqing, Zhuo

2012-09-01

A MOSFET-based electrostatic discharge (ESD) power clamp circuit with only a 10 ns RC time constant for a 0.18-μm process is proposed. A diode-connected NMOSFET is used to maintain a long delay time and save area. The special structure overcomes other shortcomings in this clamp circuit. Under fast power-up events, the gate voltage of the clamp MOSFET does not rise as quickly as under ESD events, the special structure can keep the clamp MOSFET thoroughly off. Under a falsely triggered event, the special structure can turn off the clamp MOSFET in a short time. The clamp circuit can also reject the power supply noise effectively. Simulation results show that the clamp circuit avoids fast false triggering events such as a 30 ns/1.8 V power-up, maintains a 1.2 μs delay time and a 2.14 μs turn-off time, and reduces to about 70% of the RC time constant. It is believed that the proposed clamp circuit can be widely used in high-speed integrated circuits.

Dephosphorylation of GluN2B C-Terminal Tyrosine Residues Does Not Contribute to Acute Ethanol Inhibition of Recombinant NMDA Receptors

PubMed Central

Hughes, Benjamin A.; Smothers, Corigan T.; Woodward, John J.

2013-01-01

N-methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10–600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors. PMID:23357553

A structural and mechanistic study of π-clamp-mediated cysteine perfluoroarylation.

PubMed

Dai, Peng; Williams, Jonathan K; Zhang, Chi; Welborn, Matthew; Shepherd, James J; Zhu, Tianyu; Van Voorhis, Troy; Hong, Mei; Pentelute, Bradley L

2017-08-11

Natural enzymes use local environments to tune the reactivity of amino acid side chains. In searching for small peptides with similar properties, we discovered a four-residue π-clamp motif (Phe-Cys-Pro-Phe) for regio- and chemoselective arylation of cysteine in ribosomally produced proteins. Here we report mutational, computational, and structural findings directed toward elucidating the molecular factors that drive π-clamp-mediated arylation. We show the significance of a trans conformation prolyl amide bond for the π-clamp reactivity. The π-clamp cysteine arylation reaction enthalpy of activation (ΔH ‡ ) is significantly lower than a non-π-clamp cysteine. Solid-state NMR chemical shifts indicate the prolyl amide bond in the π-clamp motif adopts a 1:1 ratio of the cis and trans conformation, while in the reaction product Pro3 was exclusively in trans. In two structural models of the perfluoroarylated product, distinct interactions at 4.7 Å between Phe1 side chain and perfluoroaryl electrophile moiety are observed. Further, solution 19 F NMR and isothermal titration calorimetry measurements suggest interactions between hydrophobic side chains in a π-clamp mutant and the perfluoroaryl probe. These studies led us to design a π-clamp mutant with an 85-fold rate enhancement. These findings will guide us toward the discovery of small reactive peptides to facilitate abiotic chemistry in water.

Solutions for transients in arbitrarily branching cables: III. Voltage clamp problems.

PubMed

Major, G

1993-07-01

Branched cable voltage recording and voltage clamp analytical solutions derived in two previous papers are used to explore practical issues concerning voltage clamp. Single exponentials can be fitted reasonably well to the decay phase of clamped synaptic currents, although they contain many underlying components. The effective time constant depends on the fit interval. The smoothing effects on synaptic clamp currents of dendritic cables and series resistance are explored with a single cylinder + soma model, for inputs with different time courses. "Soma" and "cable" charging currents cannot be separated easily when the soma is much smaller than the dendrites. Subtractive soma capacitance compensation and series resistance compensation are discussed. In a hippocampal CA1 pyramidal neurone model, voltage control at most dendritic sites is extremely poor. Parameter dependencies are illustrated. The effects of series resistance compound those of dendritic cables and depend on the "effective capacitance" of the cell. Plausible combinations of parameters can cause order-of-magnitude distortions to clamp current waveform measures of simulated Schaeffer collateral inputs. These voltage clamp problems are unlikely to be solved by the use of switch clamp methods.

Sodium efflux from voltage clamped squid giant axons.

PubMed Central

Landowne, D

1977-01-01

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

PubMed

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

Oxidation of the N-terminal methionine of lens alpha-A crystallin

NASA Technical Reports Server (NTRS)

Takemoto, L.; Horwitz, J.; Emmons, T.; Spooner, B. S. (Principal Investigator)

1992-01-01

Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different seropositive peptides (i.e. T1a and T1b) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide T1b has a molecular weight 16 atomic mass units larger than T1a, suggesting posttranslational modification. Analysis of ionization fragments of the T1b peptide by mass spectrometry demonstrates that this difference in molecular weight is due to the in vivo oxidation of the N-terminal met residue of the alpha-A crystallin molecule.

Clinical aspects of incorporating cord clamping into stabilisation of preterm infants.

PubMed

Knol, Ronny; Brouwer, Emma; Vernooij, Alex S N; Klumper, Frans J C M; DeKoninck, Philip; Hooper, Stuart B; Te Pas, Arjan B

2018-04-21

Fetal to neonatal transition is characterised by major pulmonary and haemodynamic changes occurring in a short period of time. In the international neonatal resuscitation guidelines, comprehensive recommendations are available on supporting pulmonary transition and delaying clamping of the cord in preterm infants. Recent experimental studies demonstrated that the pulmonary and haemodynamic transition are intimately linked, could influence each other and that the timing of umbilical cord clamping should be incorporated into the respiratory stabilisation. We reviewed the current knowledge on how to incorporate cord clamping into stabilisation of preterm infants and the physiological-based cord clamping (PBCC) approach, with the infant's transitional status as key determinant of timing of cord clamping. This approach could result in optimal timing of cord clamping and has the potential to reduce major morbidities and mortality in preterm infants. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Visual Associative Learning in Restrained Honey Bees with Intact Antennae

PubMed Central

Dobrin, Scott E.; Fahrbach, Susan E.

2012-01-01

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee. PMID:22701575

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

PubMed

Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

2011-09-22

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

Resin-assisted Enrichment of N-terminal Peptides for Characterizing Proteolytic Processing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jong Seo; Dai, Ziyu; Aryal, Uma K.

2013-06-17

Proteolytic processing is a ubiquitous, irreversible posttranslational modification that plays an important role in cellular regulation in all living organisms. Herein we report a resin-assisted positive selection method for specifically enriching protein N-terminal peptides to facilitate the characterization of proteolytic processing events by liquid chromatography-tandem mass spectrometry. In this approach, proteins are initially reduced and alkylated and their lysine residues are converted to homoarginines. Then, protein N-termini are selectively converted to reactive thiol groups. We demonstrate that these sequential reactions were achieved with nearly quantitative efficiencies. Thiol-containing N-terminal peptides are then captured (>98% efficiency) by a thiol-affinity resin, a significantmore » improvement over the traditional avidin/biotin enrichment. Application to cell lysates of Aspergillus niger, a filamentous fungus of interest for biomass degradation, enabled the identification of 1672 unique protein N-termini and proteolytic cleavage sites from 690 unique proteins.« less

An ideal clamping analysis for a cross-ply laminate

NASA Technical Reports Server (NTRS)

Valisetty, R. R.; Murthy, P. L. N.; Rehfield, L. W.

1988-01-01

Different elementary clamping models are discussed for a three layer crossply laminate to study the sensitivity of clamping to the definition of cross-sectional rotation. All of these models leave a considerable residual warping at the edges. Using a complimentary energy principle and principle of superposition, an analysis is conducted to reduce this residual warping. This led to the identification of exact interior solution corresponding to the ideal clamping. This study also suggests a presence of stress singularities at the corners and between different layers near the fixed edge.

High-speed pressure clamp.

PubMed

Besch, Stephen R; Suchyna, Thomas; Sachs, Frederick

2002-10-01

We built a high-speed, pneumatic pressure clamp to stimulate patch-clamped membranes mechanically. The key control element is a newly designed differential valve that uses a single, nickel-plated piezoelectric bending element to control both pressure and vacuum. To minimize response time, the valve body was designed with minimum dead volume. The result is improved response time and stability with a threefold decrease in actuation latency. Tight valve clearances minimize the steady-state air flow, permitting us to use small resonant-piston pumps to supply pressure and vacuum. To protect the valve from water contamination in the event of a broken pipette, an optical sensor detects water entering the valve and increases pressure rapidly to clear the system. The open-loop time constant for pressure is 2.5 ms for a 100-mmHg step, and the closed-loop settling time is 500-600 micros. Valve actuation latency is 120 micros. The system performance is illustrated for mechanically induced changes in patch capacitance.

Analysis of impactor residues in tray clamps from the Long Duration Exposure Facility. Part 2: Clamps from Bay B of the satellite

NASA Technical Reports Server (NTRS)

Bernhard, Ronald P.; Zolensky, Michael E.

1994-01-01

The Long Duration Exposure Facility (LDEF) was placed in low-Earth orbit (LEO) in 1984 and recovered 5.7 years later. The LDEF was host to several individual experiments specifically designed to characterize critical aspects of meteoroid and debris environment in LEO. However, it was realized from the beginning that the most efficient use of the satellite would be to examine the entire surface for impact features. In this regard, particular interest centered on common exposed materials that faced in all LDEF pointing directions. Among the most important of these materials was the tray clamps. Therefore, in an effort to better understand the nature of particulates in LEO and their effects on spacecraft hardware, residues found in impact features on LDEF tray clamp surfaces are being analyzed. This catalog presents all data from clamps from Bay B of the LDEF. NASA Technical Memorandum 104759 has cataloged impacts that occurred on Bay B (published March 1993). Subsequent catalogs will include clamps from succeeding bays of the satellite.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7.

PubMed

Edamatsu, M

2016-02-11

Kinesin-5 plays an essential role in spindle formation and function, and serves as a potential target for anti-cancer drugs. The aim of this study was to elucidate the molecular properties of the N-terminal extension of the Schizosaccharomyces pombe kinesin-5, Cut7. This extension is rich in charged amino acids and predicted to be intrinsically disordered. In S. pombe cells, a Cut7 construct lacking half the N-terminal extension failed to localize along the spindle microtubules and formed a monopolar spindle. However, a construct lacking the entire N-terminal extension exhibited normal localization and formed a typical bipolar spindle. In addition, in vitro analyses revealed that the truncated Cut7 constructs demonstrated similar motile velocities and directionalities as the wild-type motor protein, but the microtubule landing rates were significantly reduced. These findings suggest that the N-terminal extension is not required for normal Cut7 intracellular localization or function, but alters the microtubule-binding properties of this protein in vitro.

Imaging the Impact of Proton Irradiation on Edge Terminations in Vertical GaN pin Diodes

DOE PAGES

Collins, Kimberlee C.; King, Michael P.; Dickerson, Jeramy R.; ...

2017-05-29

Devices based on GaN have shown great promise for high power electronics, including their potential use as radiation tolerant components. An important step to realizing high power diodes is the design and implementation of an edge termination to mitigate field crowding, which can lead to premature breakdown. However, little is known about the effects of radiation on edge termination functionality. We experimentally examine the effects of proton irradiation on multiple field ring edge terminations in high power vertical GaN pin diodes using in operando imaging with electron beam induced current (EBIC). We find that exposure to proton irradiation influences fieldmore » spreading in the edge termination as well as carrier transport near the anode. By using depth-dependent EBIC measurements of hole diffusion length in homoepitaxial n-GaN we demonstrate that the carrier transport effect is due to a reduction in hole diffusion length following proton irradiation.« less

Imaging the Impact of Proton Irradiation on Edge Terminations in Vertical GaN pin Diodes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, Kimberlee C.; King, Michael P.; Dickerson, Jeramy R.

Devices based on GaN have shown great promise for high power electronics, including their potential use as radiation tolerant components. An important step to realizing high power diodes is the design and implementation of an edge termination to mitigate field crowding, which can lead to premature breakdown. However, little is known about the effects of radiation on edge termination functionality. We experimentally examine the effects of proton irradiation on multiple field ring edge terminations in high power vertical GaN pin diodes using in operando imaging with electron beam induced current (EBIC). We find that exposure to proton irradiation influences fieldmore » spreading in the edge termination as well as carrier transport near the anode. By using depth-dependent EBIC measurements of hole diffusion length in homoepitaxial n-GaN we demonstrate that the carrier transport effect is due to a reduction in hole diffusion length following proton irradiation.« less

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

PubMed

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Orthorexic and restrained eating behaviour in vegans, vegetarians, and individuals on a diet.

PubMed

Barthels, Friederike; Meyer, Frank; Pietrowsky, Reinhard

2018-04-01

Orthorexic eating behaviour, restrained eating, and veganism/vegetarianism are food selection strategies sharing several characteristics. Since there are no studies investigating their interrelationships, aim of the present study was to analyse orthorexic and restrained eating behaviour in (1) a sample of vegans and vegetarians and (2) a sample of individuals on a diet to lose weight. Division of samples according to pre-defined criteria in (1) vegans (n = 114), vegetarians (n = 63), individuals with rare meat consumption (n = 83) and individuals with frequent meat consumption (n = 91) and in (2) participants on a diet with dietary change (n = 104), without dietary change (n = 37) and a control group of individuals not on a diet (n = 258). Orthorexic eating behaviour was assessed with the Düsseldorfer Orthorexie Skala and restrained eating was assessed with the Restraint Eating Scale. Vegans and vegetarians do not differ in orthorexic eating behaviour, but both groups score higher in orthorexic eating behaviour than individuals consuming red meat. There are no differences regarding restrained eating. Individuals on a diet with dietary change score higher in both orthorexic and restrained eating, than individuals without dietary change and individuals not on a diet. Individuals who restrict their eating behaviour, either predominantly due to ethical reasons or with the intention to lose weight, display more orthorexic eating behaviour than individuals not limiting their food consumption. Further research is needed to investigate whether veganism, vegetarianism, or frequent dieting behaviour serve as risk factors for orthorexia. Level V, cross-sectional descriptive study.

Body Dissatisfaction Mediates the Relationship between Self-Esteem and Restrained Eating in Female Undergraduates

ERIC Educational Resources Information Center

Gianini, Loren M.; Smith, Jane Ellen

2008-01-01

The purpose of the current study was to examine the eating behavior, self-esteem, and social anxiety of restrained and non-restrained eaters exposed to an interpersonal stressor. Sixty female undergraduate students completed questionnaires and took part in a stressor and taste test. Results indicated that self-esteem was not predictive of eating…

Structure of the Tropomyosin Overlap Complex from Chicken Smooth Muscle: Insight into the Diversity of N-Terminal Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frye, Jeremiah; Klenchin, Vadim A.; Rayment, Ivan

Tropomyosin is a stereotypical {alpha}-helical coiled coil that polymerizes to form a filamentous macromolecular assembly that lies on the surface of F-actin. The interaction between the C-terminal and N-terminal segments on adjacent molecules is known as the overlap region. We report here two X-ray structures of the chicken smooth muscle tropomyosin overlap complex. A novel approach was used to stabilize the C-terminal and N-terminal fragments. Globular domains from both the human DNA ligase binding protein XRCC4 and bacteriophage {phi}29 scaffolding protein Gp7 were fused to 37 and 28 C-terminal amino acid residues of tropomyosin, respectively, whereas the 29 N-terminal aminomore » acids of tropomyosin were fused to the C-terminal helix bundle of microtubule binding protein EB1. The structures of both the XRCC4 and Gp7 fusion proteins complexed with the N-terminal EB1 fusion contain a very similar helix bundle in the overlap region that encompasses {approx}15 residues. The C-terminal coiled coil opens to allow formation of the helix bundle, which is stabilized by hydrophobic interactions. These structures are similar to that observed in the NMR structure of the rat skeletal overlap complex [Greenfield, N. J., et al. (2006) J. Mol. Biol. 364, 80-96]. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin show significant curvature, which differs somewhat between the two structures and implies flexibility in the overlap complex, at least in solution. This is likely an important attribute that allows tropomyosin to assemble around the actin filaments. These structures provide a molecular explanation for the role of N-acetylation in the assembly of native tropomyosin.« less

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency.

PubMed

Rudhe, Charlotta; Clifton, Rachel; Whelan, James; Glaser, Elzbieta

2002-12-06

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.

Dynamic Clamp in Cardiac and Neuronal Systems Using RTXI

PubMed Central

Ortega, Francis A.; Butera, Robert J.; Christini, David J.; White, John A.; Dorval, Alan D.

2016-01-01

The injection of computer-simulated conductances through the dynamic clamp technique has allowed researchers to probe the intercellular and intracellular dynamics of cardiac and neuronal systems with great precision. By coupling computational models to biological systems, dynamic clamp has become a proven tool in electrophysiology with many applications, such as generating hybrid networks in neurons or simulating channelopathies in cardiomyocytes. While its applications are broad, the approach is straightforward: synthesizing traditional patch clamp, computational modeling, and closed-loop feedback control to simulate a cellular conductance. Here, we present two example applications: artificial blocking of the inward rectifier potassium current in a cardiomyocyte and coupling of a biological neuron to a virtual neuron through a virtual synapse. The design and implementation of the necessary software to administer these dynamic clamp experiments can be difficult. In this chapter, we provide an overview of designing and implementing a dynamic clamp experiment using the Real-Time eXperiment Interface (RTXI), an open- source software system tailored for real-time biological experiments. We present two ways to achieve this using RTXI’s modular format, through the creation of a custom user-made module and through existing modules found in RTXI’s online library. PMID:25023319

Proteolytic interconversion and N-terminal sequences of the Citrobacter diversus major beta-lactamases.

PubMed Central

Franceschini, N; Amicosante, G; Perilli, M; Maccarrone, M; Oratore, A; van Beeumen, J; Frère, J M

1991-01-01

The N-terminal sequences of the two major beta-lactamases produced by Citrobacter diversus differed only by the absence of the first residue in form II and the loss of five amino acid residues at the C-terminal end. Limited proteolysis of the homogeneous form I protein yielded a variety of enzymatically active products. In the major product obtained after the action of papain, the first three N-terminal residues of form I had been cleaved, whereas at the C-terminal end the treated enzyme lacked five residues. However, this cannot explain the different behaviours of form I, form II and papain digestion product upon chromatofocusing. Form I, which was sequenced up to position 56, exhibited a very high degree of similarity with a Klebsiella oxytoca beta-lactamase. The determined sequence, which contained the active serine residue, demonstrated that the chromosome-encoded beta-lactamase of Citrobacter diversus belong to class A. Images Fig. 2. PMID:2039443

Temperature-Controlled Clamping and Releasing Mechanism

NASA Technical Reports Server (NTRS)

Rosing, David; Ford, Virginia

2005-01-01

A report describes the development of a mechanism that automatically clamps upon warming and releases upon cooling between temperature limits of approx. =180 K and approx. =293 K. The mechanism satisfied a need specific to a program that involved repeated excursions of a spectrometer between a room-temperature atmospheric environment and a cryogenic vacuum testing environment. The mechanism was also to be utilized in the intended application of the spectrometer, in which the spectrometer would be clamped for protection during launch of a spacecraft and released in the cold of outer space to allow it to assume its nominal configuration for scientific observations. The mechanism is passive in the sense that its operation does not depend on a control system and does not require any power other than that incidental to heating and cooling. The clamping and releasing action is effected by bolt-preloaded stacks of shape-memory-alloy (SMA) cylinders. In designing this mechanism, as in designing other, similar SMA mechanisms, it was necessary to account for the complex interplay among thermal expansion, elastic and inelastic deformation under load, and SMA thermomechanical properties.

Miro's N-Terminal GTPase Domain Is Required for Transport of Mitochondria into Axons and Dendrites

PubMed Central

Babic, Milos; Russo, Gary J.; Wellington, Andrea J.; Sangston, Ryan M.; Gonzalez, Migdalia

2015-01-01

Mitochondria are dynamically transported in and out of neuronal processes to maintain neuronal excitability and synaptic function. In higher eukaryotes, the mitochondrial GTPase Miro binds Milton/TRAK adaptor proteins linking microtubule motors to mitochondria. Here we show that Drosophila Miro (dMiro), which has previously been shown to be required for kinesin-driven axonal transport, is also critically required for the dynein-driven distribution of mitochondria into dendrites. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to determine the significance of dMiro's N-terminal and C-terminal GTPase domains, respectively. Expression of dMiroT25N in the absence of endogenous dMiro caused premature lethality and arrested development at a pupal stage. dMiroT25N accumulated mitochondria in the soma of larval motor and sensory neurons, and prevented their kinesin-dependent and dynein-dependent distribution into axons and dendrites, respectively. dMiroT25N mutant mitochondria also were severely fragmented and exhibited reduced kinesin and dynein motility in axons. In contrast, dMiroT460N did not impair viability, mitochondrial size, or the distribution of mitochondria. However, dMiroT460N reduced dynein motility during retrograde mitochondrial transport in axons. Finally, we show that substitutions analogous to the constitutively active Ras-G12V mutation in dMiro's N-terminal and C-terminal GTPase domains cause neomorphic phenotypic effects that are likely unrelated to the normal function of each GTPase domain. Overall, our analysis indicates that dMiro's N-terminal GTPase domain is critically required for viability, mitochondrial size, and the distribution of mitochondria out of the neuronal soma regardless of the employed motor, likely by promoting the transition from a stationary to a motile state. PMID:25855186

Chloroplast downsizing under nitrate nutrition restrained mesophyll conductance and photosynthesis in rice (Oryza sativa L.) under drought conditions.

PubMed

Li, Yong; Ren, Binbin; Yang, Xiuxia; Xu, Guohua; Shen, Qirong; Guo, Shiwei

2012-05-01

The phenomenon whereby ammonium enhances the tolerance of rice seedlings (Oryza sativa L., cv. 'Shanyou 63' hybrid indica China) to water stress has been reported in previous studies. To study the intrinsic mechanism of biomass synthesis related to photosynthesis, hydroponic experiments supplying different nitrogen (N) forms were conducted; water stress was simulated by the addition of polyethylene glycol. Water stress decreased leaf water potential (Ψ(leaf)) under nitrate nutrition, while it had no negative effect under ammonium nutrition. The decreased Ψ(leaf) under nitrate nutrition resulted in chloroplast downsizing and subsequently decreased mesophyll conductance to CO(2) (g(m)). The decreased g(m) and stomatal conductance (g(s)) under nitrate nutrition with water stress restrained the CO(2) supply to the chloroplast and Rubisco. The relatively higher distribution of leaf N to Rubisco under ammonium nutrition might also be of benefit for photosynthesis under water stress. In conclusion, chloroplast downsizing induced a decline in g(m), a relatively higher decrease in g(s) under nitrate nutrition with water stress, restrained the CO(2) supply to Rubisco and finally decreased the photosynthetic rate.

c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis

DTIC Science & Technology

2015-03-01

1 AWARD NUMBER: W81XWH-12-1-0431 TITLE: “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Sclerosis ” PRINCIPAL...TITLE AND SUBTITLE “c-jun-N-Terminal Kinase (JNK) for the Treatment of Amyotrophic Lateral Scelerosis” 5a. CONTRACT NUMBER 5b. GRANT NUMBER... Lateral   Sclerosis ”   Final  Report:  Project  Period  Sept  2012-­‐Dec  2014     Personnel  List:     Feng,  Yangbo

Comparison of oxygen saturation values obtained from fingers on physically restrained or unrestrained sides of the body.

PubMed

Korhan, Esra Akin; Yönt, Gülendam Hakverdioğlu; Khorshid, Leyla

2011-01-01

The aim of this study was to compare semiexperimentally the pulse oximetry values obtained from a finger on restrained or unrestrained sides of the body. The pulse oximeter provides a noninvasive measurement of the oxygen saturation of hemoglobin in arterial blood. One of the procedures most frequently applied to patients in intensive care units is the application of physical restraint. Circulation problems are the most important complication in patients who are physically restrained. Evaluation of oxygen saturation from body parts in which circulation is impeded or has deteriorated can cause false results. The research sample consisted of 30 hospitalized patients who participated in the study voluntarily and who were concordant with the inclusion criteria of the study. Patient information and patient follow-up forms were used for data collection. Pulse oximetry values were measured simultaneously using OxiMax Nellcor finger sensors from fingers on the restrained and unrestrained sides of the body. Numeric and percentile distributions were used in evaluating the sociodemographic properties of patients. A significant difference was found between the oxygen saturation values obtained from a finger of an arm that had been physically restrained and a finger of an arm that had not been physically restrained. The mean oxygen saturation value measured from a finger of an arm that had been physically restrained was found to be 93.40 (SD, 2.97), and the mean oxygen saturation value measured from a finger of an arm that had not been physically restrained was found to be 95.53 (SD, 2.38). The results of this study indicate that nurses should use a finger of an arm that is not physically restrained when evaluating oxygen saturation values to evaluate them correctly.

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

PubMed

Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

2015-12-01

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

PubMed

Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

2013-06-07

In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording

PubMed Central

Kolb, Ilya; Kodandaramaiah, Suhasa B.; Chubykin, Alexander A.; Yang, Aimei; Bear, Mark F.; Boyden, Edward S.; Forest, Craig R.

2014-01-01

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation. PMID:25429119

Robotic partial nephrectomy with selective parenchymal compression (Simon clamp).

PubMed

Castillo, O A; Rodriguez-Carlin, A; Lopez-Fontana, G; Aleman, E

2013-01-01

To present our initial experience using selective renal parenchymal ischemia, without hilar clamping, in robotic-assisted partial nephrectomy. In four patients with T1a renal tumor we performed robotic-assisted partial nephrectomy, using the Simon's clamp (Aesculap). It provides selective parenchymal compression without the need of vascular clamping. All patients had exofitic renal tumors in polar location. Renal parenchymal reconstruction was done as the standard technique. The median age was 49.6 years (42-59), 3 male and 1 female patient. Median operative time was 71,6 minutes (40-120). Mean stimated bleeding was 250 ml (50-400). Average tumor size was 3,25 cm (1,5-5,3). There were no complications and the average hospital stay was 3,5 days (1-7). The pathology was informed as renal cell carcinoma in three patients and one hemorrhagic cyst. The surgical margins were negative. Our preliminary results shows that selective renal parenchymal compression, with the Simon's clamp, provides an alternative to vascular control in selected patients with polar renal tumors. Copyright © 2012 AEU. Published by Elsevier Espana. All rights reserved.

U.S. Transit Track Restraining Rail. Volume II : Guidelines.

DOT National Transportation Integrated Search

1981-12-01

This report covers a study of restraining rails in transit track, which is part of the current research program of UMTA and was initiated: (1) to assist in the analysis, design, and maintenance and operation of transit track; (2) to compile guideline...

PARTIAL RESTRAINING FORCE INTRODUCTION METHOD FOR DESIGNING CONSTRUCTION COUNTERMESURE ON ΔB METHOD

NASA Astrophysics Data System (ADS)

Nishiyama, Taku; Imanishi, Hajime; Chiba, Noriyuki; Ito, Takao

Landslide or slope failure is a three-dimensional movement phenomenon, thus a three-dimensional treatment makes it easier to understand stability. The ΔB method (simplified three-dimensional slope stability analysis method) is based on the limit equilibrium method and equals to an approximate three-dimensional slope stability analysis that extends two-dimensional cross-section stability analysis results to assess stability. This analysis can be conducted using conventional spreadsheets or two-dimensional slope stability computational software. This paper describes the concept of the partial restraining force in-troduction method for designing construction countermeasures using the distribution of the restraining force found along survey lines, which is based on the distribution of survey line safety factors derived from the above-stated analysis. This paper also presents the transverse distributive method of restraining force used for planning ground stabilizing on the basis of the example analysis.

On the statistical equivalence of restrained-ensemble simulations with the maximum entropy method

PubMed Central

Roux, Benoît; Weare, Jonathan

2013-01-01

An issue of general interest in computer simulations is to incorporate information from experiments into a structural model. An important caveat in pursuing this goal is to avoid corrupting the resulting model with spurious and arbitrary biases. While the problem of biasing thermodynamic ensembles can be formulated rigorously using the maximum entropy method introduced by Jaynes, the approach can be cumbersome in practical applications with the need to determine multiple unknown coefficients iteratively. A popular alternative strategy to incorporate the information from experiments is to rely on restrained-ensemble molecular dynamics simulations. However, the fundamental validity of this computational strategy remains in question. Here, it is demonstrated that the statistical distribution produced by restrained-ensemble simulations is formally consistent with the maximum entropy method of Jaynes. This clarifies the underlying conditions under which restrained-ensemble simulations will yield results that are consistent with the maximum entropy method. PMID:23464140

[Analysis of brain hemometabolism behavior during carotid endarterectomy with temporary clamping.].

PubMed

Duval Neto, Gastão Fernandes; Niencheski, Augusto H

2004-04-01

Carotid endarterectomy with temporary clamping changes cerebral blood flow and cerebral metabolic oxygen demand ratio with consequent oligemic hypoxia or hemometabolic uncoupling. This study aimed at identifying changes in brain hemometabolism, evaluated through changes in oxyhemoglobin saturation in internal jugular vein bulb (SvjO2) during carotid endarterectomy with clamping, and at correlating these changes with potentially interfering factors, mainly end tidal CO2 pressure (P ET CO2) and cerebral perfusion pressure (CPP). Sixteen patients with unilateral carotid stenotic disease scheduled to carotid endarterectomy with carotid arterial clamping were enrolled in this study. Parameters including internal jugular bulb oxyhemoglobin saturation, stump pressure and end tidal CO2 pressure were measured at the following moments: M1 - pre-clamping; M2 - 3 minutes after clamping; M3 - pre-unclamping; M4 - post-unclamping). The comparison among SvjO2 (%, mean +/- SD) in all studied periods has shown differences between those recorded in moments M1 (52.25 +/- 7.87) and M2 (47.43 +/- 9.19). This initial decrease stabilized during temporary clamping, showing decrease in the comparison between M2 and M3 (46.56 +/- 9.25), without statistical significance (p = ns). At post-unclamping, M4 (47.68 +/- 9.12), SvjO2 was increased as compared to M2 and M3 clamping stages, however it was still lower than that of pre-clamping stage M1.(M4 x M1 - p < 0.04) This SvjO2 decrease was followed by significant cerebral perfusion pressure (stump pressure) decrease. Factors influencing this brain hemometabolic uncoupling trend were correlated to P ET CO2. The comparison between CPP and SvjO2 showed weak correlation devoid of statistical significance. In the conditions of our study, SvjO2 measurement is a fast and effective way of clinically monitoring changes in CBF/CMRO2 ratio. Temporary carotid clamping implies in a trend towards brain hemometabolic uncoupling and, as a consequence, to

Neural correlates of restrained eaters' high susceptibility to food cues: An fMRI study.

PubMed

Wang, Yu; Dong, Debo; Todd, Jackson; Du, Jie; Yang, Zhou; Lu, Hui; Chen, Hong

2016-09-19

Many studies have reported that specific susceptibility to food cues plays an important role in disordered eating behavior. However, whether restraint status modulates the neural bases of attentional bias to different types of food cues remains unknown. Thus, functional magnetic resonance imaging (fMRI) was conducted in individuals (12 restraint eaters, 12 unrestraint eaters) exposed to high/low-energy food and neutral images while performing a two-choice oddball task. The results indicated that restrained eaters responded more quickly to high-energy food images than to neutral and low-energy food images. More notably, compared with unrestrained eaters, restrained eaters showed faster reaction times, hyper-activation in a much wider array of reward (e.g., insula/orbitofrontal cortex), attention (superior frontal gyrus) and visual processing (e.g., superior temporal gyrus) regions, and hypo-activation in cognitive control areas (e.g., anterior cingulate) in response to high-energy food cues. Furthermore, among restrained eaters, the longest reaction times were found for low-energy food images, and activation of the attention and visual-related cortex (e.g., superior parietal gyrus) in the low-neutral contrast condition was significantly stronger than in unrestrained eaters. These findings contribute to our understanding of susceptibility to food cues: in addition to the special sensitivity (attentional bias) to high-energy food images, restrained eaters may also be more sensitive (allocate more attentional resources) to low-energy food images. These potential neural bases of restrained eaters may help clarify why dieting to lose or maintain weight is so often unsuccessful. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The TDP-43 N-terminal domain structure at high resolution.

PubMed

Mompeán, Miguel; Romano, Valentina; Pantoja-Uceda, David; Stuani, Cristiana; Baralle, Francisco E; Buratti, Emanuele; Laurents, Douglas V

2016-04-01

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete (1) H, (15) N and (13) C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an α-helix and six β-strands. Two β-strands form a β-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol(-1) , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol(-1) ). The β-strands, α-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the (15) N{(1) H}NOE, as well as the translational and transversal relaxation rates. Structural data have been deposited in the Protein Data Bank under accession code: 2n4p. The NMR assignments have been deposited in the BMRB database under access code: 25675. © 2016 Federation of European Biochemical Societies.

Glutamic Acid as a Precursor to N-Terminal Pyroglutamic Acid in Mouse Plasmacytoma Protein

PubMed Central

Twardzik, Daniel R.; Peterkofsky, Alan

1972-01-01

Cell suspensions derived from a mouse plasmacytoma (RPC-20) that secretes an immunoglobulin light chain containing N-terminal pyroglutamic acid can synthesize protein in vitro. Chromatographic examination of an enzymatic digest of protein labeled with glutamic acid shows only labeled glutamic acid and pyroglutamic acid; hydrolysis of protein from cells labeled with glutamine, however, yields substantial amounts of glutamic acid in addition to glutamine and pyroglutamic acid. The absence of glutamine synthetase and presence of glutaminase in plasmacytoma homogenates is consistent with these findings. These data indicate that N-terminal pyroglutamic acid can be derived from glutamic acid without prior conversion of glutamic acid to glutamine. Since free or bound forms of glutamine cyclize nonezymatically to pyroglutamate with ease, while glutamic acid does not, the data suggest that N-terminal pyroglutamic acid formation from glutamic acid is enzymatic rather than spontaneous. Images PMID:4400295

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

NASA Astrophysics Data System (ADS)

Webber, Nathaniel; He, Yi; Reilly, James P.

2014-02-01

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m2 and m2+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

System and Apparatus for Deploying a Satellite

NASA Technical Reports Server (NTRS)

Santos, Luis H. (Inventor); Hudeck, John D. (Inventor)

2016-01-01

A frictionless satellite constraint system is provided. The constraint system includes at least one clamp bar configured to restrain a satellite within the constraint system in an axial direction. The constraint system also includes a plurality of pins configured to restrain the satellite within the constraint system in a lateral direction.

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

PubMed Central

Afonso, José P.; Chintakayala, Kiran; Suwannachart, Chatrudee; Sedelnikova, Svetlana; Giles, Kevin; Hoyes, John B.; Soultanas, Panos; Rafferty, John B.; Oldham, Neil J.

2013-01-01

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ′ Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ′ complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ′ binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3. PMID:23525462

PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

PubMed Central

Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

2015-01-01

Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

Normally-off AlGaN/GaN-based MOS-HEMT with self-terminating TMAH wet recess etching

NASA Astrophysics Data System (ADS)

Son, Dong-Hyeok; Jo, Young-Woo; Won, Chul-Ho; Lee, Jun-Hyeok; Seo, Jae Hwa; Lee, Sang-Heung; Lim, Jong-Won; Kim, Ji Heon; Kang, In Man; Cristoloveanu, Sorin; Lee, Jung-Hee

2018-03-01

Normally-off AlGaN/GaN-based MOS-HEMT has been fabricated by utilizing damage-free self-terminating tetramethyl ammonium hydroxide (TMAH) recess etching. The device exhibited a threshold voltage of +2.0 V with good uniformity, extremely small hysteresis of ∼20 mV, and maximum drain current of 210 mA/mm. The device also exhibited excellent off-state performances, such as breakdown voltage of ∼800 V with off-state leakage current as low as ∼10-12 A and high on/off current ratio (Ion/Ioff) of 1010. These excellent device performances are believed to be due to the high quality recessed surface, provided by the simple self-terminating TMAH etching.

Novel 384-well population patch clamp electrophysiology assays for Ca2+-activated K+ channels.

PubMed

John, Victoria H; Dale, Tim J; Hollands, Emma C; Chen, Mao Xiang; Partington, Leanne; Downie, David L; Meadows, Helen J; Trezise, Derek J

2007-02-01

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Seismic rehabilitation of skewed and curved bridges using a new generation of buckling restrained braces : research brief.

DOT National Transportation Integrated Search

2016-12-01

Damage to skewed and curved bridges during strong earthquakes is documented. This project investigates whether such damage could be mitigated by using buckling restrained braces. Nonlinear models show that using buckling restrained braces to mitigate...

Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.

PubMed

Tanaka, Masafumi; Dhanasekaran, Padmaja; Nguyen, David; Ohta, Shinya; Lund-Katz, Sissel; Phillips, Michael C; Saito, Hiroyuki

2006-08-29

The tertiary structure of lipid-free apolipoprotein (apo) A-I in the monomeric state comprises two domains: a N-terminal alpha-helix bundle and a less organized C-terminal domain. This study examined how the N- and C-terminal segments of apoA-I (residues 1-43 and 223-243), which contain the most hydrophobic regions in the molecule and are located in opposite structural domains, contribute to the lipid-free conformation and lipid interaction. Measurements of circular dichroism in conjunction with tryptophan and 8-anilino-1-naphthalenesulfonic acid fluorescence data demonstrated that single (L230P) or triple (L230P/L233P/Y236P) proline insertions into the C-terminal alpha helix disrupted the organization of the C-terminal domain without affecting the stability of the N-terminal helix bundle. In contrast, proline insertion into the N terminus (Y18P) disrupted the bundle structure in the N-terminal domain, indicating that the alpha-helical segment in this region is part of the helix bundle. Calorimetric and gel-filtration measurements showed that disruption of the C-terminal alpha helix significantly reduced the enthalpy and free energy of binding of apoA-I to lipids, whereas disruption of the N-terminal alpha helix had only a small effect on lipid binding. Significantly, the presence of the Y18P mutation offset the negative effects of disruption/removal of the C-terminal helical domain on lipid binding, suggesting that the alpha helix around Y18 concealed a potential lipid-binding region in the N-terminal domain, which was exposed by the disruption of the helix-bundle structure. When these results are taken together, they indicate that the alpha-helical segment in the N terminus of apoA-I modulates the lipid-free structure and lipid interaction in concert with the C-terminal domain.

An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

PubMed Central

Rose, K; Kocher, H P; Blumberg, B M; Kolakofsky, D

1984-01-01

A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure. PMID:6421284

Term babies with delayed cord clamping: an approach in preventing anemia (.).

PubMed

Ertekin, Arif Aktug; Nihan Ozdemir, Nilufer; Sahinoglu, Zeki; Gursoy, Tugba; Erbil, Nazan; Kaya, Erdal

2016-09-01

We investigated the effects of delayed and early clamping of the cord on the hematologic status of the baby at birth and at the end of second month. Umbilical cord of 74 babies were clamped in the first 30 s (Group 1) and 76 were clamped at 90-120 s (Group 2). Levels of hemoglobin, hematocrit, iron and ferritin were analyzed from the umbilical cord blood at birth and from the venous samples at the end of second month. Hemoglobin, hematocrit, iron and ferritin levels of cord blood were similar in both groups. However, their levels other than ferritin were higher in Group 2 at the end of second month. Two babies had respiratory distress and twelve neonates received phototherapy in Group 2 whereas only five neonates received phototherapy in Group 1. Term babies to whom delayed cord clamping was performed had improved hematological parameters at the end of second month. Therefore, delaying cord clamping in these babies may be a favorible approach in preventing anemia.

Electrostatic Interactions at the Dimer Interface Stabilize the E. coli β Sliding Clamp.

PubMed

Purohit, Anirban; England, Jennifer K; Douma, Lauren G; Tondnevis, Farzaneh; Bloom, Linda B; Levitus, Marcia

2017-08-22

Sliding clamps are ring-shaped oligomeric proteins that encircle DNA and associate with DNA polymerases for processive DNA replication. The dimeric Escherichia coli β-clamp is closed in solution but must adopt an open conformation to be assembled onto DNA by a clamp loader. To determine what factors contribute to the stability of the dimer interfaces in the closed conformation and how clamp dynamics contribute to formation of the open conformation, we identified conditions that destabilized the dimer and measured the effects of these conditions on clamp dynamics. We characterized the role of electrostatic interactions in stabilizing the β-clamp interface. Increasing salt concentration results in decreased dimer stability and faster subunit dissociation kinetics. The equilibrium dissociation constant of the dimeric clamp varies with salt concentration as predicted by simple charge-screening models, indicating that charged amino acids contribute to the remarkable stability of the interface at physiological salt concentrations. Mutation of a charged residue at the interface (Arg-103) weakens the interface significantly, whereas effects are negligible when a hydrophilic (Ser-109) or a hydrophobic (Ile-305) amino acid is mutated instead. It has been suggested that clamp opening by the clamp loader takes advantage of spontaneous opening-closing fluctuations at the clamp's interface, but our time-resolved fluorescence and fluorescence correlation experiments rule out conformational fluctuations that lead to a significant fraction of open states. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Renal Vascular Clamp Placement: A Potential Cause of Incomplete Hilar Control during Partial Nephrectomy.

PubMed

Tryon, David; Myklak, Kristene; Alsyouf, Muhannad; Conceicao, Carol; Peplinski, Brandon; Arenas, Javier L; Faaborg, Daniel; Ruckle, Herbert C; Baldwin, D Duane

2016-03-01

Previous benchtop studies have shown that robotic bulldog clamps provide incomplete vascular control of a Penrose drain. We determined the efficacy of robotic and laparoscopic bulldog clamps to ensure hemostasis on the human renal artery. The effect of clamp position on vascular control was also examined. Fresh human cadaveric renal arteries were used to determine the leak point pressure of 7 bulldog clamps from a total of 3 manufacturers. Five trials were performed per clamp at 4 locations, including the fulcrum, proximal, middle and distal positions. Comparison was done using the Kruskal-Wallis test with p <0.05 considered significant. None of the bulldog clamps leaked at a pressure less than 215 mm Hg when applied at the proximal, middle or distal position. In general leak point pressure decreased as the artery was positioned more distal along the clamp. The exception was when the vessel was placed at the fulcrum position. At that position 80% to 100% of trials with the Klein laparoscopic, 100% with the Klein robotic (Klein Robotic, San Antonio, Texas) and 60% to 80% with the Scanlan robotic (Scanlan International, Saint Paul, Minnesota) clamp leaked at pressure below 215 mm Hg. Each vascular clamp adequately occluded flow at physiological pressure when placed at the proximal, middle or distal position. Furthermore, these results demonstrate that there is leakage at physiological pressure when the artery is placed at the fulcrum of certain clamp types. These results suggest that applying a bulldog clamp at the fulcrum could potentially lead to inadequate vessel occlusion and intraoperative bleeding. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

A retrospective analysis of laparoscopic partial nephrectomy with segmental renal artery clamping and factors that predict postoperative renal function.

PubMed

Li, Pu; Qin, Chao; Cao, Qiang; Li, Jie; Lv, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Tang, Lijun; Shao, Pengfei

2016-10-01

To evaluate the feasibility and efficiency of laparoscopic partial nephrectomy (LPN) with segmental renal artery clamping, and to analyse the factors affecting postoperative renal function. We conducted a retrospective analysis of 466 consecutive patients undergoing LPN using main renal artery clamping (group A, n = 152) or segmental artery clamping (group B, n = 314) between September 2007 and July 2015 in our department. Blood loss, operating time, warm ischaemia time (WIT) and renal function were compared between groups. Univariable and multivariable linear regression analyses were applied to assess the correlations of selected variables with postoperative glomerular filtration rate (GFR) reduction. Volumetric data and estimated GFR of a subset of 60 patients in group B were compared with GFR to evaluate the correlation between these functional variables and preserved renal function after LPN. The novel technique slightly increased operating time, WIT and intra-operative blood loss (P < 0.001), while it provided better postoperative renal function (P < 0.001) compared with the conventional technique. The blocking method and tumour characteristics were independent factors affecting GFR reduction, while WIT was not an independent factor. Correlation analysis showed that estimated GFR presented better correlation with GFR compared with kidney volume (R(2) = 0.794 cf. R(2) = 0.199) in predicting renal function after LPN. LPN with segmental artery clamping minimizes warm ischaemia injury and provides better early postoperative renal function compared with clamping the main renal artery. Kidney volume has a significantly inferior role compared with eGFR in predicting preserved renal function. © 2016 The Authors BJU International © 2016 BJU International Published by John Wiley & Sons Ltd.

A Dynamic Clamp on Every Rig

PubMed Central

2017-01-01

Abstract The dynamic clamp should be a standard part of every cellular electrophysiologist’s toolbox. That it is not, even 25 years after its introduction, comes down to three issues: money, the disruption that adding dynamic clamp to an existing electrophysiology rig entails, and the technical prowess required of experimenters. These have been valid and limiting issues in the past, but no longer. Technological advances associated with the so-called maker movement render them moot. We demonstrate this by implementing a fast (∼100 kHz) dynamic clamp system using an inexpensive microcontroller (Teensy 3.6). The overall cost of the system is less than USD$100, and assembling it requires no prior electronics experience. Modifying it—for example, to add Hodgkin–Huxley-style conductances—requires no prior programming experience. The system works together with existing electrophysiology data acquisition systems (for Macintosh, Windows, and Linux); it does not attempt to supplant them. Moreover, the process of assembling, modifying, and using the system constitutes a useful pedagogical exercise for students and researchers with no background but an interest in electronics and programming. We demonstrate the system’s utility by implementing conductances as fast as a transient sodium conductance and as complex as the Ornstein–Uhlenbeck conductances of the “point conductance” model of synaptic background activity. PMID:29085905

A twin study of differences in the response of plasma ghrelin to a milkshake preload in restrained eaters

PubMed Central

Myhre, Rachel; Kratz, Mario; Goldberg, Jack; Polivy, Janet; Melhorn, Susan; Buchwald, Dedra; Cummings, David E.; Schur, Ellen A.

2014-01-01

Background Genetic, physiological, and psychological factors can affect food intake, but twin studies can distinguish inherited from environmental contributors. We examined the influence of attempted cognitive control of eating (“restrained eating”) on levels of appetite-regulating hormones. Methods Sixteen female, monozygotic twin pairs, discordant for Restraint Scale score (i.e., one twin a restrained eater with score > 15 whereas the co-twin was unrestrained), were selected from the University of Washington Twin Registry. Serial plasma ghrelin concentrations were monitored during meals and a preload study paradigm involving intake of a milkshake followed by an ad libitum ice cream “taste test.” Results Body weight, body mass index, resting energy expenditure, and fasting leptin levels were very similar between restrained and unrestrained twins. In a preload study, twins ate similar amounts of ice cream shortly after drinking identical milkshakes (mean ±SD; restrained 239 ±158 vs. unrestrained 228 ±132 kilocalories; P = 0.83). However, ghrelin concentrations during the preload study were significantly higher (P = 0.03) in restrained twins than in their unrestrained co-twins. Regardless of restraint status, ghrelin levels prior to the preload study were prospectively and positively associated with ice cream intake (P = 0.001). Conclusions Compared to their unrestrained co-twins, restrained twins had higher endogenous ghrelin levels during a preload study, but ate similar amounts. This finding is consistent with exertion of cognitive control relative to the state of physiologic appetite stimulation. Moreover, these findings in twins suggest that higher ghrelin levels result from restrained eating behavior and not from genetic predisposition. PMID:24534168

Rapid exhumation of Cretaceous arc-rocks along the Blue Mountains restraining bend of the Enriquillo-Plantain Garden fault, Jamaica, using thermochronometry from multiple closure systems

NASA Astrophysics Data System (ADS)

Cochran, William J.; Spotila, James A.; Prince, Philip S.; McAleer, Ryan J.

2017-11-01

The effect of rapid erosion on kinematic partitioning along transpressional plate margins is not well understood, particularly in highly erosive climates. The Blue Mountains restraining bend (BMRB) of eastern Jamaica, bound to the south by the left-lateral Enriquillo-Plantain Garden fault (EPGF), offers an opportunity to test the effects of highly erosive climatic conditions on a 30-km-wide restraining bend system. No previous thermochronometric data exists in Jamaica to describe the spatial or temporal pattern of rock uplift and how oblique (> 20°) plate motion is partitioned into vertical strain. To define the exhumation history, we measured apatite (n = 10) and zircon (n = 6) (U-Th)/He ages, 40Ar/39Ar (n = 2; amphibole and K-spar) ages, and U/Pb zircon (n = 2) crystallization ages. Late Cretaceous U/Pb and 40Ar/39Ar ages (74-68 Ma) indicate rapid cooling following shallow emplacement of plutons during north-south subduction along the Great Caribbean Arc. Early to middle Miocene zircon helium ages (19-14 Ma) along a vertical transect suggest exhumation and island emergence at 0.2 mm/yr. Older zircon ages 10-15 km to the north (44-35 Ma) imply less rock uplift. Apatite helium ages are young (6-1 Ma) across the entire orogen, suggesting rapid exhumation of the BMRB since the late Miocene. These constraints are consistent with previous reports of restraining bend formation and early emergence of eastern Jamaica. An age-elevation relationship from a vertical transect implies an exhumation rate of 0.8 mm/yr, while calculated closure depths and thermal modeling suggests exhumation as rapid as 2 mm/yr. The rapid rock uplift rates in Jamaica are comparable to the most intense transpressive zones worldwide, despite the relatively slow (5-7 mm/yr) strike-slip rate. We hypothesize highly erosive conditions in Jamaica enable a higher fraction of plate motion to be accommodated by vertical deformation. Thus, strike-slip restraining bends may evolve differently depending on

Rapid exhumation of Cretaceous arc-rocks along the Blue Mountains restraining bend of the Enriquillo-Plantain Garden fault, Jamaica, using thermochronometry from multiple closure systems

USGS Publications Warehouse

Cochran, William J.; Spotila, James A.; Prince, Philip S.; McAleer, Ryan J.

2017-01-01

The effect of rapid erosion on kinematic partitioning along transpressional plate margins is not well understood, particularly in highly erosive climates. The Blue Mountains restraining bend (BMRB) of eastern Jamaica, bound to the south by the left-lateral Enriquillo-Plantain Garden fault (EPGF), offers an opportunity to test the effects of highly erosive climatic conditions on a 30-km-wide restraining bend system. No previous thermochronometric data exists in Jamaica to describe the spatial or temporal pattern of rock uplift and how oblique (> 20°) plate motion is partitioned into vertical strain. To define the exhumation history, we measured apatite (n = 10) and zircon (n = 6) (U-Th)/He ages, 40Ar/39Ar (n = 2; amphibole and K-spar) ages, and U/Pb zircon (n = 2) crystallization ages. Late Cretaceous U/Pb and 40Ar/39Ar ages (74–68 Ma) indicate rapid cooling following shallow emplacement of plutons during north-south subduction along the Great Caribbean Arc. Early to middle Miocene zircon helium ages (19–14 Ma) along a vertical transect suggest exhumation and island emergence at ~ 0.2 mm/yr. Older zircon ages 10–15 km to the north (44–35 Ma) imply less rock uplift. Apatite helium ages are young (6–1 Ma) across the entire orogen, suggesting rapid exhumation of the BMRB since the late Miocene. These constraints are consistent with previous reports of restraining bend formation and early emergence of eastern Jamaica. An age-elevation relationship from a vertical transect implies an exhumation rate of 0.8 mm/yr, while calculated closure depths and thermal modeling suggests exhumation as rapid as 2 mm/yr. The rapid rock uplift rates in Jamaica are comparable to the most intense transpressive zones worldwide, despite the relatively slow (5–7 mm/yr) strike-slip rate. We hypothesize highly erosive conditions in Jamaica enable a higher fraction of plate motion to be accommodated by vertical deformation. Thus, strike-slip restraining bends may evolve differently

In-Situ-measurement of restraining forces during forming of rectangular cups

NASA Astrophysics Data System (ADS)

Singer, M.; Liewald, M.

2016-11-01

This contribution introduces a new method for evaluating the restraining forces during forming of rectangular cups with the goal of eliminating the disadvantages of the currently used scientifically established measurement procedures. With this method forming forces are measured indirectly by the elastic deformation of die structure caused by locally varying tribological system. Therefore, two sensors were integrated into the punch, which measure the restraining forces during the forming process. Furthermore, it was possible to evaluate the effects of different lubricants showing the time dependent trend as a function of stroke during the forming of the materials DP600 and DC04. A main advantage of this testing method is to get real friction corresponding data out of the physical deep drawing process as well as the measurement of real acting restraining forces at different areas of the deep drawing part by one single test. Measurement results gained by both sensors have been integrated into LS-Dyna simulation in which the coefficient of friction was regarded as a function of time. The simulated and deep drawn parts afterwards are analysed and compared to specific areas with regard to locally measured thickness of part. Results show an improvement of simulation quality when using locally varying, time dependent coefficients of friction compared to commonly used constant values.

Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

PubMed Central

Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

2000-01-01

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

PubMed Central

Das, Debasis; Krantz, Bryan A.

2016-01-01

Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins—protective antigen (PA), lethal factor (LF), and edema factor—translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity. PMID:27506790

30 CFR 77.603 - Clamping of trailing cables to equipment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Clamping of trailing cables to equipment. 77.603 Section 77.603 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL... UNDERGROUND COAL MINES Trailing Cables § 77.603 Clamping of trailing cables to equipment. Trailing cables...

The effect of aliskiren on urinary cytokine/chemokine responses to clamped hyperglycaemia in type 1 diabetes.

PubMed

Cherney, David Z I; Reich, Heather N; Scholey, James W; Daneman, Denis; Mahmud, Farid H; Har, Ronnie L H; Sochett, Etienne B

2013-10-01

Acute clamped hyperglycaemia activates the renin-angiotensin-aldosterone system (RAAS) and increases the urinary excretion of inflammatory cytokines/chemokines in patients with uncomplicated type 1 diabetes mellitus. Our objective was to determine whether blockade of the RAAS would blunt the effect of acute hyperglycaemia on urinary cytokine/chemokine excretion, thereby giving insights into potentially protective effects of these agents prior to the onset of clinical nephropathy. Blood pressure, renal haemodynamic function (inulin and para-aminohippurate clearances) and urinary cytokines/chemokines were measured after 6 h of clamped euglycaemia (4-6 mmol/l) and hyperglycaemia (9-11 mmol/l) on two consecutive days in patients with type 1 diabetes mellitus (n = 27) without overt nephropathy. Measurements were repeated after treatment with aliskiren (300 mg daily) for 30 days. Before aliskiren, clamped hyperglycaemia increased filtration fraction (from 0.188 ± 0.007 to 0.206 ± 0.007, p = 0.003) and urinary fibroblast growth factor-2 (FGF2), IFN-α2 and macrophage-derived chemokine (MDC) (p < 0.005). After aliskiren, the filtration fraction response to hyperglycaemia was abolished, resulting in a lower filtration fraction after aliskiren under clamped hyperglycaemic conditions (p = 0.004), and none of the biomarkers increased in response to hyperglycaemia. Aliskiren therapy also reduced levels of urinary eotaxin, FGF2, IFN-α2, IL-2 and MDC during clamped hyperglycaemia (p < 0.005). The increased urinary excretion of inflammatory cytokines/chemokines in response to acute hyperglycaemia is blunted by RAAS blockade in humans with uncomplicated type 1 diabetes mellitus.

Intraoperative brachial plexus injury during emergence following movement with arms restrained: a preventable complication?

PubMed Central

Chandler, Mark H; DiMatteo, Laura; Hasenboehler, Erik A; Temple, Michael

2007-01-01

Background Despite considerable analysis and preventive strategies, brachial plexus injuries remain fairly common in the perioperative setting. These injuries range from brief periods of numbness or discomfort in the immediate postoperative period to, in rare cases, profound, prolonged losses of sensation and function. We present a case of an orthopedic surgery patient who suffered a brachial plexus injury while under anesthesia after trying to sit upright with his arms restrained. Case presentation After the uneventful placement of an intramedullary tibial nail, an 18 year old patient tried to sit upright with his arms restrained while still under the influence of anesthesia. In the immediate postoperative period, the patient complained of a profound loss of sensation in his left arm and an inability to flex his left elbow, suppinate his arm, or abduct and rotate his shoulder. Neurological examination and subsequent studies revealed a C5-6 brachial plexus injury. The patient underwent range of motion physical therapy and, over the next three months, regained the full function and sensation of his left arm. Conclusion Restraining arms during general anesthesia to prevent injury remains a wise practice. However, to avoid injuring the brachial plexus while the arms are restrained, extra caution must be used to prevent unexpected patient movement and to ensure gentle emergence. PMID:18271944

Investigation into scatter radiation dose levels received by a restrainer in small animal radiography.

PubMed

Barber, J; McNulty, J P

2012-10-01

To measure the intensity and distribution of scatter radiation received by a restrainer in veterinary radiography including the intensity of scatter radiation passing through lead protective devices at pre-defined positions. Anthropomorphic phantoms and a Labrador dog cadaver were used to simulate a restrainer and patient. Scatter dose measurements were recorded at the position of the restraining hands, thyroid, breast and gonads with and without appropriate lead protection. This was repeated for the eight most common projections as identified in an initial retrospective survey. Manual restraint of an animal for a radiographic procedure will result in a scatter radiation dose to the restrainer. The level of radiation dose varies between body regions and between projections. The use of appropriate lead protection resulted in statistically significant dose reductions to all body regions with maximum scatter dose reductions between 93 and 100%. While the doses recorded were small (μGy) in terms of associated risk, they are nonetheless cumulative which can result in a more significant dose. Therefore manual restraint should be avoided and forms of immobilisation should be used such as mechanical means, sedation or general anaesthesia. However, if completely necessary both principles of distance and adequate lead protection should be employed. © 2012 British Small Animal Veterinary Association.

A monolithic patch-clamping amplifier with capacitive feedback.

PubMed

Prakash, J; Paulos, J J; Jensen, D N

1989-03-01

Patch-clamping is an established method for directly measuring ionic transport through cellular membranes with sufficient resolution to observe open/close transitions of individual channel molecules. This paper describes an alternative technique for patch-clamping which uses a capacitor as the transimpedance element. This approach eliminates bandwidth and saturation limitations experienced with resistive patch-clamping amplifiers. A complete monolithic design featuring an on-chip operational amplifier, a capacitor array with gain-ranging from 30 pF down to 0.03 pF, and reset and gain ranging switches has been fabricated using 5 microns CMOS technology. It is shown that the voltage noise of the CMOS operational amplifier limits the overall noise performance, but that performance competitive with conventional instruments can be achieved over a 10 kHz bandwidth, at least for small input capacitances (less than or equal to 5 pF). Results are presented along with an analysis and comparison of noise performance using both resistive and capacitive elements.

Open-access microfluidic patch-clamp array with raised lateral cell trapping sites.

PubMed

Lau, Adrian Y; Hung, Paul J; Wu, Angela R; Lee, Luke P

2006-12-01

A novel open-access microfluidic patch-clamp array chip with lateral cell trapping sites raised above the bottom plane of the chip was developed by combining both a microscale soft-lithography and a macroscale polymer fabrication method. This paper demonstrates the capability of using such an open-access fluidic system for patch-clamp measurements. The surface of the open-access patch-clamp sites prepared by the macroscale hole patterning method of soft-state elastic polydimethylsiloxane (PDMS) is examined; the seal resistances are characterized and correlated with the aperture dimensions. Whole cell patch-clamp measurements are carried out with CHO cells expressing Kv2.1 ion channels. Kv2.1 ion channel blocker (TEA) dosage response is characterized and the binding activity is examined. The results demonstrate that the system is capable of performing whole cell measurements and drug profiling in a more efficient manner than the traditional patch-clamp set-up.

Measuring beta-cell function relative to insulin sensitivity in youth: Does the hyperglycemic clamp suffice?

USDA-ARS?s Scientific Manuscript database

To compare beta-cell function relative to insulin sensitivity, disposition index (DI), calculated from two clamps (2cDI, insulin sensitivity from the hyperinsulinemic-euglycemic clamp and first-phase insulin from the hyperglycemic clamp) with the DI calculated from the hyperglycemic clamp alone (hcD...

The N-terminal sequence of albumin Redhill, a variant of human serum albumin.

PubMed

Hutchinson, D W; Matejtschuk, P

1985-12-02

Albumin Redhill, a variant human albumin, has been isolated by fast protein liquid chromatofocusing. The N-terminal sequence of this protein corresponded to that of albumin A except that one additional arginine residue was attached to the N-terminus.

Direct vascular control results in less physiologic derangement than proximal aortic clamping in a porcine model of noncompressible extrathoracic torso hemorrhage.

PubMed

White, Joseph M; Cannon, Jeremy W; Stannard, Adam; Burkhardt, Gabriel E; Spencer, Jerry R; Williams, Ken; Oh, John S; Rasmussen, Todd E

2011-11-01

The optimal method of vascular control and resuscitation in patients with life-threatening, extrathoracic torso hemorrhage remains debated. Guidelines recommend emergency department thoracotomy (EDT) with aortic clamping, although transabdominal aortic clamping followed by vascular control and direct vascular control (DVC) without aortic clamping are alternatives. The objective of this study is to compare the effectiveness of three approaches to extrathoracic torso hemorrhage in a large animal model. Adolescent swine (Sus Scrofa) (mean weight = 80.9 kg) were randomized into three groups all of which had class IV shock established by hemorrhage from an iliac artery injury. Group 1: EDT with thoracic aortic clamping (N = 6); group 2: transabdominal supraceliac aortic clamping (SCC; N = 6); and group 3: DVC of bleeding site without aortic clamping (N = 6). After hemorrhage, EDT or SCC was performed in groups 1 and 2, respectively, with subsequent exploration of the bleeding site and placement of a temporary vascular shunt (TVS). Group 3 (DVC) underwent direct exploration of the injury and placement of a TVS. All groups were resuscitated to predefined physiologic endpoints over 6 hours with repeated measures of central and cerebral perfusion and end-organ function at standardized time points. Postmortem tissue analysis was performed to quantify injury to critical tissue beds. There was no difference in mortality among the groups and no TVS failures. Central aortic pressure, carotid flow, and partial pressure brain tissue oximetry, all demonstrated increases in EDT and SCC after application of the aortic clamp relative to DVC (p < 0.05). During resuscitation, serum lactate levels were higher in EDT compared with SCC and DVC (6.85 vs. 3.08 and 2.15, respectively; p < 0.05) and serum pH in EDT reflected greater acidosis than SCC and DVC (7.24 vs. 7.36 and 7.39, respectively; p < 0.05). EDT and SCC required more intravenous fluid than DVC (2,166 mL and 2,166 mL vs. 667 m

Specific amino acid residues in the beta sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli.

PubMed

Sutton, Mark D; Duzen, Jill M

2006-03-07

Escherichia coli dnaN159 strains encode a mutant form of the beta sliding clamp (beta159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the beta clamp with the alpha catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (beta-G174A), while the other contained D173A, G174A and H175A substitutions (beta-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of Delta polB (pol II), Delta dinB (pol IV) and/or Delta umuDC (pol V) alleles. Taken together, these findings indicate that mutations in the beta clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in beta159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.

Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells.

PubMed

Klemic, Kathryn G; Klemic, James F; Reed, Mark A; Sigworth, Fred J

2002-06-01

The patch clamp method measures membrane currents at very high resolution when a high-resistance 'gigaseal' is established between the glass microelectrode and the cell membrane (Pflugers Arch. 391 (1981) 85; Neuron 8 (1992) 605). Here we describe the first use of the silicone elastomer, poly(dimethylsiloxane) (PDMS), for patch clamp electrodes. PDMS is an attractive material for patch clamp recordings. It has low dielectric loss and can be micromolded (Annu. Rev. Mat. Sci. 28 (1998) 153) into a shape that mimics the tip of the glass micropipette. Also, the surface chemistry of PDMS may be altered to mimic the hydrophilic nature of glass (J. Appl. Polym. Sci. 14 (1970) 2499; Annu. Rev. Mat. Sci. 28 (1998) 153), thereby allowing a high-resistance seal to a cell membrane. We present a planar electrode geometry consisting of a PDMS partition with a small aperture sealed between electrode and bath chambers. We demonstrate that a planar PDMS patch electrode, after oxidation of the elastomeric surface, permits patch clamp recording on Xenopus oocytes. Our results indicate the potential for high-throughput patch clamp recording with a planar array of PDMS electrodes.

Dendrimer-assisted patch-clamp sizing of nuclear pores

PubMed Central

Bustamante, J.O.; Michelette, E.R.F.; Geibel, J.P.; Hanover, J.A.; McDonnell, T.J.; Dean, D.A.

2015-01-01

Macromolecular translocation (MMT) across the nuclear envelope (NE) occurs exclusively through the nuclear pore complex (NPC). Therefore, the diameter of the NPC aqueous/electrolytic channel (NPCC) is important for cellular structure and function. The NPCC diameter was previously determined to be ≅10 nm with electron microscopy (EM) using the translocation of colloidal gold particles. Here we present patch-clamp and fluorescence microscopy data from adult cardiomyocyte nuclei that demonstrate the use of patch-clamp for assessing NPCC diameter. Fluorescence microscopy with B-phycoerythrin (BPE, 240 kDa) conjugated to a nuclear localization signal (NLS) demonstrated that these nuclei were competent for NPC-mediated MMT (NPC-MMT). Furthermore, when exposed to an appropriate cell lysate, the nuclei expressed enhanced green fluorescence protein (EGFP) after 5–10 h of incubation with the plasmid for this protein (pEGFP, 3.1 MDa). Nucleus-attached patch-clamp showed that colloidal gold particles were not useful probes; they modified NPCC gating. As a result of this finding, we searched for an inert class of particles that could be used without irreversibly affecting NPCC gating and found that fluorescently labeled Star-burst dendrimers, a distinct class of polymers, were useful. Our patch-clamp and fluorescence microscopy data with calibrated dendrimers indicate that the cardiomyocyte NPCC diameter varies between 8 and 9 nm. These studies open a new direction in the investigation of live, continuous NPC dynamics under physiological conditions. PMID:10784359

Motor vehicle crash-related injury causation scenarios for spinal injuries in restrained children and adolescents.

PubMed

Zonfrillo, Mark R; Locey, Caitlin M; Scarfone, Steven R; Arbogast, Kristy B

2014-01-01

Motor vehicle crash (MVC)-related spinal injuries result in significant morbidity and mortality in children. The objective was to identify MVC-related injury causation scenarios for spinal injuries in restrained children. This was a case series of occupants in MVCs from the Crash Injury Research and Engineering Network (CIREN) data set. Occupants aged 0-17 years old with at least one Abbreviated Injury Scale (AIS) 2+ severity spinal injury in vehicles model year 1990+ that did not experience a rollover were included. Unrestrained occupants, those not using the shoulder portion of the belt restraint, and those with child restraint gross misuse were excluded. Occupants with preexisting comorbidities contributing to spinal injury and occupants with limited injury information were also excluded. A multidisciplinary team retrospectively reviewed each case to determine injury causation scenarios (ICSs). Crash conditions, occupant and restraint characteristics, and injuries were qualitatively summarized. Fifty-nine cases met the study inclusion criteria and 17 were excluded. The 42 occupants included sustained 97 distinct AIS 2+ spinal injuries (27 cervical, 22 thoracic, and 48 lumbar; 80 AIS-2, 15 AIS-3, 1 AIS-5, and 1 AIS-6), with fracture as the most common injury type (80%). Spinal-injured occupants were most frequently in passenger cars (64%), and crash direction was most often frontal (62%). Mean delta-V was 51.3 km/h±19.4 km/h. The average occupant age was 12.4±5.3 years old, and 48% were 16- to 17-year-olds. Thirty-six percent were right front passengers and 26% were drivers. Most occupants were lap and shoulder belt restrained (88%). Non-spinal AIS 2+ injuries included those of the lower extremity and pelvis (n=56), head (n=43), abdomen (n=39), and thorax (n=36). Spinal injury causation was typically due to flexion or lateral bending over the lap and or shoulder belt or child restraint harness, compression by occupant's own seat back, or axial loading through

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva

2014-11-01

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminusmore » is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.« less

Application of active electrode compensation to perform continuous voltage-clamp recordings with sharp microelectrodes.

PubMed

Gómez-González, J F; Destexhe, A; Bal, T

2014-10-01

Electrophysiological recordings of single neurons in brain tissues are very common in neuroscience. Glass microelectrodes filled with an electrolyte are used to impale the cell membrane in order to record the membrane potential or to inject current. Their high resistance induces a high voltage drop when passing current and it is essential to correct the voltage measurements. In particular, for voltage clamping, the traditional alternatives are two-electrode voltage-clamp technique or discontinuous single electrode voltage-clamp (dSEVC). Nevertheless, it is generally difficult to impale two electrodes in a same neuron and the switching frequency is limited to low frequencies in the case of dSEVC. We present a novel fully computer-implemented alternative to perform continuous voltage-clamp recordings with a single sharp-electrode. To reach such voltage-clamp recordings, we combine an active electrode compensation algorithm (AEC) with a digital controller (AECVC). We applied two types of control-systems: a linear controller (proportional plus integrative controller) and a model-based controller (optimal control). We compared the performance of the two methods to dSEVC using a dynamic model cell and experiments in brain slices. The AECVC method provides an entirely digital method to perform continuous recording and smooth switching between voltage-clamp, current clamp or dynamic-clamp configurations without introducing artifacts.

Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

PubMed

Kaur, Gurmeet; Subramanian, Srikrishna

2017-10-18

Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

Voltage-Clamp Studies on Uterine Smooth Muscle

PubMed Central

Anderson, Nels C.

1969-01-01

These studies have developed and tested an experimental approach to the study of membrane ionic conductance mechanisms in strips of uterine smooth muscle. The experimental and theoretical basis for applying the double sucrose-gap technique is described along with the limitations of this system. Nonpropagating membrane action potentials were produced in response to depolarizing current pulses under current-clamp conditions. The stepwise change of membrane potential under voltage-clamp conditions resulted in a family of ionic currents with voltage- and time-dependent characteristics. In sodium-free solution the peak transient current decreased and its equilibrium potential shifted along the voltage axis toward a more negative internal potential. These studies indicate a sodium-dependent, regenerative excitation mechanism. PMID:5796366

Experimental Tests of a Real Building Seismically Retrofitted by Special Buckling-Restrained Braces

NASA Astrophysics Data System (ADS)

D'Aniello, Mario; Corte, Gaetano Della; Mazzolani, Federico M.

2008-07-01

Buckling Restrained Braces (BRBs), differently from conventional braces, do not exhibit appreciable difference between the tensile and compression capacity and no strength degradation of brace capacity under compressive and cyclic loading. Since lateral and local buckling behaviour modes are restrained, large inelastic capacities are attainable. Hence, BRBs may represent an efficient and reliable solution for reducing the seismic vulnerability of buildings. Results of experimental tests on the response of a real two-story reinforced concrete (RC) building equipped with BRBs are presented and discussed. The considered BRBs are a special `only-steel' version of the more common `unbonded braces'. In particular, two different BRBs have been tested. Both of them are detachable "only-steel" devices, consisting in a rectangular steel plate and a restraining steel sleeve. The latter is composed by two omega shapes which are bolted together. The main characteristic of the braces consists in the possibility to hide them within the space between the facing and the backing of masonry infill walls commonly used for RC buildings.

Ribonucleocapsid Formation of SARS-COV Through Molecular Action of the N-Terminal Domain of N Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.

Conserved amongst all coronaviruses are four structural proteins, the matrix (M), small envelope (E) and spike (S) that are embedded in the viral membrane and the nucleocapsid phosphoprotein (N), which exists in a ribonucleoprotein complex in their lumen. The N terminal domain of coronaviral N proteins (N-NTD) provides a scaffold for RNA binding while the C-terminal domain (N-CTD) mainly acts as oligomerization modules during assembly. The C-terminus of N protein anchors it to the viral membrane by associating with M protein. We characterized the structures of N-NTD from severe acute respiratory syndrome coronavirus (SARS-CoV) in two crystal forms, at 1.17Amore » (monoclinic) and 1.85 A (cubic) respectively, solved by molecular replacement using the homologous avian infectious bronchitis virus (IBV) structure. Flexible loops in the solution structure of SARS-CoV N-NTD are now shown to be well ordered around the beta-sheet core. The functionally important positively charged beta-hairpin protrudes out of the core and is oriented similar to that in the IBV N-NTD and is involved in crystal packing in the monoclinic form. In the cubic form, the monomers form trimeric units that stack in a helical array. Comparison of crystal packing of SARS-CoV and IBV N-NTDs suggest a common mode of RNA recognition, but probably associate differently in vivo during the formation of the ribonucleoprotein complex. Electrostatic potential distribution on the surface of homology models of related coronaviral N-NTDs hints that they employ different modes of both RNA recognition as well as oligomeric assembly, perhaps explaining why their nucleocapsids have different morphologies.« less

Protection against β-amyloid neurotoxicity by a non-toxic endogenous N-terminal β-amyloid fragment and its active hexapeptide core sequence.

PubMed

Forest, Kelly H; Alfulaij, Naghum; Arora, Komal; Taketa, Ruth; Sherrin, Tessi; Todorovic, Cedomir; Lawrence, James L M; Yoshikawa, Gene T; Ng, Ho-Leung; Hruby, Victor J; Nichols, Robert A

2018-01-01

High levels (μM) of beta amyloid (Aβ) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aβ, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aβ at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aβ. An N-terminal Aβ fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aβ-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aβ fragment and a shorter hexapeptide core sequence in the Aβ fragment (Aβcore: 10-15) to protect or reverse Aβ-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aβ fragment and Aβcore on Aβ-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aβ fragment and Aβcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aβcore were also shown to be fully protective against Aβ-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aβ fragment, while active stabilized N-terminal Aβcore derivatives offer the potential for therapeutic application. © 2017 International Society for Neurochemistry.

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Basic Research Program, SAIC-Frederick, Frederick, MD 21702; Gustchina, Alla

2010-08-01

The medium-resolution structure of the N-terminal fragment of E. coli Lon protease shows that this part of the enzyme consists of two compact domains and a very long α-helix. The structure of a recombinant construct consisting of residues 1–245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 Å resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very longmore » C-terminal α-helix. The structure of the first subdomain (residues 1–117), which consists mostly of β-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas the second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

A nitric oxide concentration clamp.

PubMed

Zhelyaskov, V R; Godwin, D W

1999-10-01

We report a new method of generating nitric oxide (NO) that possesses several advantages for experimental use. This method consists of a photolysis chamber where NO is released by illuminating photolabile NO donors with light from a xenon lamp, in conjunction with feedback control. Control of the photolysis light was achieved by selectively gating light projected through a shutter before the light was launched into a light guide that conveyed the light to the photolysis chamber. By gating the light in proportion to a sensor that reported nearly instantaneous concentration from the photolysis chamber, a criterion NO concentration could be achieved, which could be easily adjusted to higher or lower criterion levels. To denote the similarity of this process with the electrophysiological process of voltage clamp, we term this process a concentration "clamp." This development enhances the use of the fiber-optic-based system for NO delivery and should enable the execution of experiments where the in situ concentration of NO is particularly critical, such as in biological preparations. Copyright 1999 Academic Press.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

PubMed

Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

2004-06-01

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

Missense Mutations in the N-Terminal Domain of Human Phenylalanine Hydroxylase Interfere with Binding of Regulatory Phenylalanine

PubMed Central

Gjetting, Torben; Petersen, Marie; Guldberg, Per; Güttler, Flemming

2001-01-01

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46–48 (GAL) and 65–69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2–120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency. PMID:11326337

Rediscovering sperm ion channels with the patch-clamp technique

PubMed Central

Kirichok, Yuriy; Lishko, Polina V.

2011-01-01

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca2+ in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca2+ and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible. PMID:21642646

Effects of Imperfect Dynamic Clamp: Computational and Experimental Results

PubMed Central

Bettencourt, Jonathan C.; Lillis, Kyle P.; White, John A.

2008-01-01

In the dynamic clamp technique, a typically nonlinear feedback system delivers electrical current to an excitable cell that represents the actions of “virtual” ion channels (e.g., channels that are gated by local membrane potential or by electrical activity in neighboring biological or virtual neurons). Since the conception of this technique, there have been a number of different implementations of dynamic clamp systems, each with differing levels of flexibility and performance. Embedded hardware-based systems typically offer feedback that is very fast and precisely timed, but these systems are often expensive and sometimes inflexible. PC-based systems, on the other hand, allow the user to write software that defines an arbitrarily complex feedback system, but real-time performance in PC-based systems can be deteriorated by imperfect real-time performance. Here we systematically evaluate the performance requirements for artificial dynamic clamp knock-in of transient sodium and delayed rectifier potassium conductances. Specifically we examine the effects of controller time step duration, differential equation integration method, jitter (variability in time step), and latency (the time lag from reading inputs to updating outputs). Each of these control system flaws is artificially introduced in both simulated and real dynamic clamp experiments. We demonstrate that each of these errors affect dynamic clamp accuracy in a way that depends on the time constants and stiffness of the differential equations being solved. In simulations, time steps above 0.2 ms lead to catastrophic alteration of spike shape, but the frequency-vs.-current relationship is much more robust. Latency (the part of the time step that occurs between measuring membrane potential and injecting re-calculated membrane current) is a crucial factor as well. Experimental data are substantially more sensitive to inaccuracies than simulated data. PMID:18076999

Natural Vibration Analysis of Clamped Rectangular Orthotropic Plates

NASA Astrophysics Data System (ADS)

dalaei, m.; kerr, a. d.

The natural vibrations of clamped rectangular orthotropic plates are analyzed using the extended Kantorovich method. The developed iterative scheme converges very rapidly to the final result. The obtained natural frequencies are evaluated for a square plate made of Kevlar 49 Epoxy and the obtained results are compared with those published by Kanazawa and Kawai, and by Leissa. The agreement was found to be very close. As there are no exact analytical solutions for clamped rectangular plates, the generated closed form expression for the natural modes, and the corresponding natural frequencies, are very suitable for use in engineering analyses.

Temporary clamping of external carotid artery in convexity, parasagittal and temporal base meningioma.

PubMed

Yadav, Yad Ram; Parihar, Vijay; Agarwal, Moneet; Bhatele, Pushp Raj

2012-01-01

The management of intraoperative bleeding during removal of a large hyper vascular meningioma is crucial for safe and efficient surgery. Preoperative embolization of meningioma is the best way to reduce vascularity of meningiomas but this technique is not readily available, costly and has its own limitations. The study is aimed to evaluate the use of temporary clamping of external carotid artery to reduce blood loss and operating time during excision of large convexity, parasagittal or temporal base meningiomas. A prospective study of 115 consecutively operated meningiomas of size 5 cms or more were operated from January 2002 to December2010. Temporary clamping of external carotid artery was done in 61 while 51 cases were managed without clamping. There was significant reduction of blood loss, operative time and blood transfusion given in the temporary clipping group compared to non clipping group. There was stitch abscess in two patients each in clamping, and non clamping group. There was no scalp necrosis or mortality in any of the group. Temporary clamping of external carotid artery is a safe, simple and cost-effective alternative to embolization for the surgery of large meningiomas. This can be practiced at all the centers.

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

NASA Technical Reports Server (NTRS)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Experimental and numerical analysis of clamped joints in front motorbike suspensions

NASA Astrophysics Data System (ADS)

Croccolo, D.; de Agostinis, M.; Vincenzi, N.

2010-06-01

Clamped joints are shaft-hub connections used, as an instance, in front motorbike suspensions to lock the steering plates with the legs and the legs with the wheel pin, by means of one or two bolts. The preloading force, produced during the tightening process, should be evaluated accurately, since it must lock safely the shaft, without overcoming the yielding point of the hub. Firstly, friction coefficients have been evaluated on “ad-hoc designed” specimens, by applying the Design of Experiment approach: the applied tightening torque has been precisely related to the imposed preloading force. Then, the tensile state of clamps have been evaluated both via FEM and by leveraging some design formulae proposed by the Authors as function of the preloading force and of the clamp geometry. Finally, the results have been compared to those given by some strain gauges applied on the tested clamps: the discrepancies between numerical analyses, the design formulae and the experimental results remains under a threshold of 10%.

Analysis of motion during the breast clamping phase of mammography

PubMed Central

McEntee, Mark F; Mercer, Claire; Kelly, Judith; Millington, Sara; Hogg, Peter

2016-01-01

Objective: To measure paddle motion during the clamping phase of a breast phantom for a range of machine/paddle combinations. Methods: A deformable breast phantom was used to simulate a female breast. 12 mammography machines from three manufacturers with 22 flexible and 20 fixed paddles were evaluated. Vertical motion at the paddle was measured using two calibrated linear potentiometers. For each paddle, the motion in millimetres was recorded every 0.5 s for 40 s, while the phantom was compressed with 80 N. Independent t-tests were used to determine differences in paddle motion between flexible and fixed, small and large, GE Senographe Essential (General Electric Medical Systems, Milwaukee, WI) and Hologic Selenia Dimensions paddles (Hologic, Bedford, MA). Paddle tilt in the medial–lateral plane for each machine/paddle combination was calculated. Results: All machine/paddle combinations demonstrate highest levels of motion during the first 10 s of the clamping phase. The least motion is 0.17 ± 0.05 mm/10 s (n = 20) and the most motion is 0.51 ± 0.15 mm/10 s (n = 80). There is a statistical difference in paddle motion between fixed and flexible (p < 0.001), GE Senographe Essential and Hologic Selenia Dimensions paddles (p < 0.001). Paddle tilt in the medial–lateral plane is independent of time and varied from 0.04 ° to 0.69 °. Conclusion: All machine/paddle combinations exhibited motion and tilting, and the extent varied with machine and paddle sizes and types. Advances in knowledge: This research suggests that image blurring will likely be clinically insignificant 4 s or more after the clamping phase commences. PMID:26739577

Formation of pyroglutamic acid from N-terminal glutamic acid in immunoglobulin gamma antibodies.

PubMed

Chelius, Dirk; Jing, Kay; Lueras, Alexis; Rehder, Douglas S; Dillon, Thomas M; Vizel, Alona; Rajan, Rahul S; Li, Tiansheng; Treuheit, Michael J; Bondarenko, Pavel V

2006-04-01

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

Synthesis of 3-iodoindoles by the Pd/Cu-catalyzed coupling of N,N-dialkyl-2-iodoanilines and terminal acetylenes, followed by electrophilic cyclization.

PubMed

Yue, Dawei; Yao, Tuanli; Larock, Richard C

2006-01-06

[reaction: see text] 3-Iodoindoles have been prepared in excellent yields by coupling terminal acetylenes with N,N-dialkyl-o-iodoanilines in the presence of a Pd/Cu catalyst, followed by an electrophilic cyclization of the resulting N,N-dialkyl-o-(1-alkynyl)anilines using I2 in CH2Cl2. Aryl-, vinylic-, alkyl-, and silyl-substituted terminal acetylenes undergo this process to produce excellent yields of 3-iodoindoles. The reactivity of the carbon-nitrogen bond cleavage during cyclization follows the following order: Me > n-Bu, Me > Ph, and cyclohexyl > Me. Subsequent palladium-catalyzed Sonogashira, Suzuki, and Heck reactions of the resulting 3-iodoindoles proceed smoothly in good yields.

29 CFR 101.37 - Application for temporary relief or restraining orders.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROCEDURES Procedure Under Section 10 (j) and (l) of the Act § 101.37 Application for temporary relief or restraining orders. Whenever it is deemed advisable to seek temporary injunctive relief under section 10(j) or...

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal.

PubMed

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, Δ espF ), N-terminal sequence (219 bp, Δ espF N ), and C-terminal sequence (528 bp, Δ espF C ) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, Δ espF/pespF , Δ espF N /pespF N , and Δ espF C /pespF C by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), Δ espF , Δ espF/pespF , Δ espF C , Δ espF C /pespF C , Δ espF N , and Δ espF N /pespF N groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, Δ espF/pespF , and Δ espF C were significantly higher than that of Δ espF , Δ espF N , Δ espF C /pespF C , and Δ espF N /pespF N group ( p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain.

Determination of the pKa of the N-terminal amino group of ubiquitin by NMR

PubMed Central

Oregioni, Alain; Stieglitz, Benjamin; Kelly, Geoffrey; Rittinger, Katrin; Frenkiel, Tom

2017-01-01

Ubiquitination regulates nearly every aspect of cellular life. It is catalysed by a cascade of three enzymes and results in the attachment of the C-terminal carboxylate of ubiquitin to a lysine side chain in the protein substrate. Chain extension occurs via addition of subsequent ubiquitin molecules to either one of the seven lysine residues of ubiquitin, or via its N-terminal α-amino group to build linear ubiquitin chains. The pKa of lysine side chains is around 10.5 and hence E3 ligases require a mechanism to deprotonate the amino group at physiological pH to produce an effective nucleophile. In contrast, the pKa of N-terminal α-amino groups of proteins can vary significantly, with reported values between 6.8 and 9.1, raising the possibility that linear chain synthesis may not require a general base. In this study we use NMR spectroscopy to determine the pKa for the N-terminal α-amino group of methionine1 of ubiquitin for the first time. We show that it is 9.14, one of the highest pKa values ever reported for this amino group, providing a rational for the observed need for a general base in the E3 ligase HOIP, which synthesizes linear ubiquitin chains. PMID:28252051

Incremental update of electrostatic interactions in adaptively restrained particle simulations.

PubMed

Edorh, Semeho Prince A; Redon, Stéphane

2018-04-06

The computation of long-range potentials is one of the demanding tasks in Molecular Dynamics. During the last decades, an inventive panoply of methods was developed to reduce the CPU time of this task. In this work, we propose a fast method dedicated to the computation of the electrostatic potential in adaptively restrained systems. We exploit the fact that, in such systems, only some particles are allowed to move at each timestep. We developed an incremental algorithm derived from a multigrid-based alternative to traditional Fourier-based methods. Our algorithm was implemented inside LAMMPS, a popular molecular dynamics simulation package. We evaluated the method on different systems. We showed that the new algorithm's computational complexity scales with the number of active particles in the simulated system, and is able to outperform the well-established Particle Particle Particle Mesh (P3M) for adaptively restrained simulations. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Preparation of Drosophila central neurons for in situ patch clamping.

PubMed

Ryglewski, Stefanie; Duch, Carsten

2012-10-15

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker(1,2). Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain(3,4) and ventral nerve cord of embryonic(5,6), larval(7,8,9,10), and adult Drosophila(11,12,13,14). A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN5(15)), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique

Watching your weight? The relations between watching soaps and music television and body dissatisfaction and restrained eating in young girls.

PubMed

Anschutz, Doeschka; Engels, Rutger; Leeuwe, Jan Van; Strien, Tatjana van

2009-11-01

Although previous research showed that the thin ideal provided by the media affects body image and eating behaviour in young children, less is known about specific media contents that are related to body image and eating behaviour. This study tested the associations between watching soaps and music television and body dissatisfaction and restrained eating directly, and indirectly through thin ideal internalisation. We conducted a survey in class, in which 245 girls (aged 7-9) completed scales on their television watching behaviour, thin ideal internalisation, body dissatisfaction and restrained eating. Additionally, height and weight were measured. Watching soaps and music television often was associated with higher thin ideal internalisation, which in turn was associated with higher body dissatisfaction and restrained eating. Furthermore, a direct association between watching soaps and music television and restrained eating was found. If watching other types of children's programmes or maternal encouragement to be thin were included in the models, watching soaps and music television remained an important factor, especially with regard to restrained eating. Therefore, our results suggest that if young girls watch soaps and music television often, this is related to higher restrained eating and body dissatisfaction, directly or indirectly, through higher thin ideal internalisation.

Altered frontal inter-hemispheric resting state functional connectivity is associated with bulimic symptoms among restrained eaters.

PubMed

Chen, Shuaiyu; Dong, Debo; Jackson, Todd; Su, Yanhua; Chen, Hong

2016-01-29

Theory and research have indicated that restrained eating (RE) increases risk for binge-eating and eating disorder symptoms. According to the goal conflict model, such risk may result from disrupted hedonic-feeding control and its interaction with reward-driven eating. However, RE-related alterations in functional interactions among associated underlying brain regions, especially between the cerebral hemispheres, have rarely been examined directly. Therefore, we investigated inter-hemispheric resting-state functional connectivity (RSFC) among female restrained eaters (REs) (n=23) and unrestrained eaters (UREs) (n=24) following food deprivation as well as its relation to overall bulimia nervosa (BN) symptoms using voxel-mirrored homotopic connectivity (VMHC). Seed-based RSFC associated with areas exhibiting significant VMHC differences was also assessed. Compared to UREs, REs showed reduced VMHC in the dorsal-lateral prefrontal cortex (DLPFC), an area involved in inhibiting hedonic overeating. REs also displayed decreased RSFC between the right DLPFC and regions associated with reward estimation--the ventromedial prefrontal cortex (VMPFC) and posterior cingulate cortex (PCC). Finally, bulimic tendencies had a negative correlation with VMHC in the DLPFC and a positive correlation with functional connectivity (DLPFC and VMPFC) among REs but not UREs. Findings suggested that reduced inter-hemispheric functional connectivity in appetite inhibition regions and altered functional connectivity in reward related regions may help to explain why some REs fail to control hedonically-motivated feeding and experience higher associated levels of BN symptomatology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

PubMed Central

Kim, Jin S.; Nanfara, Michael T.; Chodavarapu, Sundari; Jin, Kyeong S.; Babu, Vignesh M. P.; Ghazy, Mohamed A.; Chung, Scisung

2017-01-01

Abstract Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda–sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda–β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda–β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda–β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. PMID:28168278

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination

PubMed Central

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-01-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals. PMID:23024214

A multipronged strategy of an anti-terminator protein to overcome Rho-dependent transcription termination.

PubMed

Muteeb, Ghazala; Dey, Debashish; Mishra, Saurabh; Sen, Ranjan

2012-12-01

One of the important role of Rho-dependent transcription termination in bacteria is to prevent gene expressions from the bacteriophage DNA. The transcription anti-termination systems of the lambdoid phages have been designed to overcome this Rho action. The anti-terminator protein N has three interacting regions, which interact with the mRNA, with the NusA and with the RNA polymerase. Here, we show that N uses all these interaction modules to overcome the Rho action. N and Rho co-occupy their overlapping binding sites on the nascent RNA (the nutR/tR1 site), and this configuration slows down the rate of ATP hydrolysis and the rate of RNA release by Rho from the elongation complex. N-RNA polymerase interaction is not too important for this Rho inactivation process near/at the nutR site. This interaction becomes essential when the elongation complex moves away from the nutR site. From the unusual NusA-dependence property of a Rho mutant E134K, a suppressor of N, we deduced that the N-NusA complex in the anti-termination machinery reduces the efficiency of Rho by removing NusA from the termination pathway. We propose that NusA-remodelling is also one of the mechanisms used by N to overcome the termination signals.

Design and control of a novel gastroscope intervention mechanism with circumferentially pneumatic-driven clamping function.

PubMed

Li, Yanmin; Liu, Hao; Hao, Siwen; Li, Hongyi; Han, Jianda; Yang, Yunsheng

2017-03-01

Robot-assisted manipulation is promising for solving problems such as understaffing and the risk of infection in gastro-intestinal endoscopy. However, the commonly used friction rollers in few existing systems have a potential risk of deforming flexible endoscopes for non-uniform clamping. This paper presents a robotic system for a standard flexible endoscope and focuses on a novel gastroscope intervention mechanism (GIM), which provides circumferentially uniform clamping with an airbag. The GIM works with a relay-on mechanism in a way similar to manual operation. The shear stiffness of airbag and the critical slipping force (CSF) were analysed to determine the parameters of the airbag. A fuzzy PID controller was employed to realize a fast response and high accuracy of pneumatic actuation. Experiments were performed to evaluate the accuracy, stiffness and CSF. In vitro and in vivo animal experiments were also carried out. The GIM realized an accuracy of 0.025 ± 0.2 mm and -0.03 ± 0.25° for push-pull and rotation without delivery resistance. Under < 10 N delivery resistance, the error caused by the airbag stiffness was < 0.24 mm. A quadratic polynomial could be used to describe the relationship between the CSF and pneumatic pressure. The novel GIM could effectively deliver gastroscopes. The pneumatic-driven clamping method proposed could protect the gastroscope by circumferentially uniform clamping force and the CSF could be properly controlled to guarantee operating safety. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Different methods of hilar clamping during partial nephrectomy: Impact on renal function.

PubMed

Lee, Jeong Woo; Kim, Hwanik; Choo, Minsoo; Park, Yong Hyun; Ku, Ja Hyeon; Kim, Hyeon Hoe; Kwak, Cheol

2014-03-01

To evaluate the impact of different hilar clamping methods on changes in renal function after partial nephrectomy. We analyzed the clinical data of 369 patients who underwent partial nephrectomy for a single renal tumor of size ≤4.0 cm and a normal contralateral kidney. Patients were separated into three groups depending on hilar clamping method: non-clamping, cold ischemia and warm ischemia. Estimated glomerular filtration rate was examined at preoperative, nadir and 1 year postoperatively. Percent change in estimated glomerular filtration rate was used as the parameter to assess the renal functional outcome. Percent change in nadir estimated glomerular filtration rate in the non-clamping group was significantly less compared with the cold ischemia and warm ischemia groups (P < 0.001). However, no significant differences among the groups were noted in percent change of estimated glomerular filtration rate at 1 year (P = 0.348). The cold ischemia group had a similar serial change of postoperative renal function compared with the warm ischemia group. Percent change in 1-year estimated glomerular filtration rate increased with increasing ischemia time in the cold ischemia (P for trend = 0.073) and warm ischemia groups (P for trend = 0.010). On multivariate analysis, hilar clamping (both warm ischemia and cold ischemia) were significantly associated with percent change in nadir estimated glomerular filtration rate, but not in 1-year estimated glomerular filtration rate. Non-clamping partial nephrectomy results in a lower percent change in nadir estimated glomerular filtration rate, whereas it carries an estimated glomerular filtration rate change at 1 year that is similar to partial nephrectomy with cold ischemia and warm ischemia. Cold ischemia and warm ischemia provide a similar effect on renal function. Therefore, when hilar clamping is required, minimization of ischemia time is necessary. © 2013 The Japanese Urological Association.

Make up your mind about food: A healthy mindset attenuates attention for high-calorie food in restrained eaters.

PubMed

Werthmann, Jessica; Jansen, Anita; Roefs, Anne

2016-10-01

Attention bias for food could be a cognitive pathway to overeating in obesity and restrained eating. Yet, empirical evidence for individual differences (e.g., in restrained eating and body mass index) in attention bias for food is mixed. We tested experimentally if temporarily induced health versus palatability mindsets influenced attention bias for food, and whether restrained eating moderated this relation. After manipulating mindset (health vs. palatability) experimentally, food-related attention bias was measured by eye-movements (EM) and response latencies (RL) during a visual probe task depicting high-calorie food and non-food. Restrained eating was assessed afterwards. A significant interaction of mindset and restrained eating on RL bias emerged, β = 0.36, t(58) = 2.05, p = 0.045: A health mindset - as compared to a palatability mindset - attenuated attention bias for high-caloric food only in participants with higher eating restraint. No effects were observed on EM biases. The current results demonstrate that state differences in health versus palatability mindsets can cause attenuated attention bias for high-calorie food cues in participants with higher eating restraint. Our findings add to emerging evidence that state differences in mindsets can bias attention for food, above the influence of trait differences. Copyright © 2016. Published by Elsevier Ltd.

Implementation of a fast 16-Bit dynamic clamp using LabVIEW-RT.

PubMed

Kullmann, Paul H M; Wheeler, Diek W; Beacom, Joshua; Horn, John P

2004-01-01

The dynamic-clamp method provides a powerful electrophysiological tool for creating virtual ionic conductances in living cells and studying their influence on membrane potential. Here we describe G-clamp, a new way to implement a dynamic clamp using the real-time version of the Lab-VIEW programming environment together with a Windows host, an embedded microprocessor that runs a real-time operating system and a multifunction data-acquisition board. The software includes descriptions of a fast voltage-dependent sodium conductance, delayed rectifier, M-type and A-type potassium conductances, and a leak conductance. The system can also read synaptic conductance waveforms from preassembled data files. These virtual conductances can be reliably implemented at speeds < or =43 kHz while simultaneously saving two channels of data with 16-bit precision. G-clamp also includes utilities for measuring current-voltage relations, synaptic strength, and synaptic gain. Taking an approach built on a commercially available software/hardware platform has resulted in a system that is easy to assemble and upgrade. In addition, the graphical programming structure of LabVIEW should make it relatively easy for others to adapt G-clamp for new experimental applications.

Self-terminated etching of GaN with a high selectivity over AlGaN under inductively coupled Cl2/N2/O2 plasma with a low-energy ion bombardment

NASA Astrophysics Data System (ADS)

Zhong, Yaozong; Zhou, Yu; Gao, Hongwei; Dai, Shujun; He, Junlei; Feng, Meixin; Sun, Qian; Zhang, Jijun; Zhao, Yanfei; DingSun, An; Yang, Hui

2017-10-01

Etching of GaN/AlGaN heterostructure by O-containing inductively coupled Cl2/N2 plasma with a low-energy ion bombardment can be self-terminated at the surface of the AlGaN layer. The estimated etching rates of GaN and AlGaN were 42 and 0.6 nm/min, respectively, giving a selective etching ratio of 70:1. To study the mechanism of the etching self-termination, detailed characterization and analyses were carried out, including X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS). It was found that in the presence of oxygen, the top surface of the AlGaN layer was converted into a thin film of (Al,Ga)Ox with a high bonding energy, which effectively prevented the underlying atoms from a further etching, resulting in a nearly self-terminated etching. This technique enables a uniform and reproducible fabrication process for enhancement-mode high electron mobility transistors with a p-GaN gate.

Tris(1,10-phenanthroline-κ2 N,N′)nickel(II) hexa­oxido-μ-peroxido-disulfate­(VI) N,N-dimethyl­formamide disolvate monohydrate

PubMed Central

Harvey, Miguel Angel; Suarez, Sebastián; Doctorovich, Fabio; Baggio, Ricardo

2013-01-01

The asymmetric unit of the title complex, [Ni(C12H8N2)3]S2O8·2C3H7NO·H2O, consists of a complex [Ni(phen)3]2+ cation and one isolated pds anion, with two DMF mol­ecules and one water mol­ecule as solvates (where phen is 1,10-phenanthroline, pds is the hexa­oxido-μ-peroxoido-di­sulf­ate dianion and DMF is dimethyl­formamide). The [Ni(phen)3]2+ cation is regular, with an almost ideal NiII bond-valence sum of 2.07 v.u. The group, as well as the water solvent mol­ecule, are well behaved in terms of crystallographic order, but the remaining three mol­ecules in the structure display different kinds of disorder, viz. the two DMF mol­ecules mimic a twofold splitting and the pds anion has both S atoms clamped at well-determined positions but with a not-too-well-defined central part. These peculiar behaviours are a consequence of the hydrogen-bonding inter­actions: the outermost SO3 parts of the pds anion are heavily connected to the complex cations via C—H⋯O hydrogen bonding, generating an [Ni(phen)3]pds network and providing for the stability of the terminal pds sites. Also, the water solvent mol­ecule is strongly bound to the structure (being a donor of two strong bonds and an acceptor of one) and is accordingly perfectly ordered. The peroxide O atoms in the pds middle region, instead, appear as much less restrained into their sites, which may explain their tendency to disorder. The cation–anion network leaves large embedded holes, amounting to about 28% of the total crystal volume, which are occupied by the DMF mol­ecules. The latter are weakly inter­acting with the rest of the structure, which renders them much more labile and, accordingly, prone to disorder. PMID:23476355

Mere exposure to palatable food cues reduces restrained eaters' physical effort to obtain healthy food.

PubMed

van Koningsbruggen, Guido M; Stroebe, Wolfgang; Aarts, Henk

2012-04-01

We examined whether exposure to cues of attractive food reduces effortful behavior toward healthy foods for restrained eaters. After manipulating food pre-exposure, we recorded handgrip force while presenting participants with pictures of healthy food objects. Because participants were led to expect that they could obtain each object (not specified beforehand) by squeezing the handgrip as forcefully as possible while the object was displayed on the screen, the recorded handgrip force constitutes a measure of spontaneous effortful behavior. Results show that restrained eaters, but not unrestrained eaters, displayed less forceful action toward healthy food objects (i.e., lower exertion of force) when pre-exposed to tempting food cues. No effects were found on palatability perceptions of the healthy foods. The results provide further insight into why restrained eaters have difficulties in maintaining a low-calorie diet in food-rich environments. Copyright © 2011 Elsevier Ltd. All rights reserved.

Acetaminophen Metabolite N-Acylphenolamine Induces Analgesia via Transient Receptor Potential Vanilloid 1 Receptors Expressed on the Primary Afferent Terminals of C-fibers in the Spinal Dorsal Horn.

PubMed

Ohashi, Nobuko; Uta, Daisuke; Sasaki, Mika; Ohashi, Masayuki; Kamiya, Yoshinori; Kohno, Tatsuro

2017-08-01

The widely used analgesic acetaminophen is metabolized to N-acylphenolamine, which induces analgesia by acting directly on transient receptor potential vanilloid 1 or cannabinoid 1 receptors in the brain. Although these receptors are also abundant in the spinal cord, no previous studies have reported analgesic effects of acetaminophen or N-acylphenolamine mediated by the spinal cord dorsal horn. We hypothesized that clinical doses of acetaminophen induce analgesia via these spinal mechanisms. We assessed our hypothesis in a rat model using behavioral measures. We also used in vivo and in vitro whole cell patch-clamp recordings of dorsal horn neurons to assess excitatory synaptic transmission. Intravenous acetaminophen decreased peripheral pinch-induced excitatory responses in the dorsal horn (53.1 ± 20.7% of control; n = 10; P < 0.01), while direct application of acetaminophen to the dorsal horn did not reduce these responses. Direct application of N-acylphenolamine decreased the amplitudes of monosynaptic excitatory postsynaptic currents evoked by C-fiber stimulation (control, 462.5 ± 197.5 pA; N-acylphenolamine, 272.5 ± 134.5 pA; n = 10; P = 0.022) but not those evoked by stimulation of Aδ-fibers. These phenomena were mediated by transient receptor potential vanilloid 1 receptors, but not cannabinoid 1 receptors. The analgesic effects of acetaminophen and N-acylphenolamine were stronger in rats experiencing an inflammatory pain model compared to naïve rats. Our results suggest that the acetaminophen metabolite N-acylphenolamine induces analgesia directly via transient receptor potential vanilloid 1 receptors expressed on central terminals of C-fibers in the spinal dorsal horn and leads to conduction block, shunt currents, and desensitization of these fibers.

Motor Vehicle Crash–Related Injury Causation Scenarios for Spinal Injuries in Restrained Children and Adolescents

PubMed Central

ZONFRILLO, MARK R.; LOCEY, CAITLIN M.; SCARFONE, STEVEN R.; ARBOGAST, KRISTY B.

2016-01-01

Objective Motor vehicle crash (MVC)-related spinal injuries result in significant morbidity and mortality in children. The objective was to identify MVC-related injury causation scenarios for spinal injuries in restrained children. Methods This was a case series of occupants in MVCs from the Crash Injury Research and Engineering Network (CIREN) data set. Occupants aged 0–17 years old with at least one Abbreviated Injury Scale (AIS) 2+ severity spinal injury in vehicles model year 1990+ that did not experience a rollover were included. Unrestrained occupants, those not using the shoulder portion of the belt restraint, and those with child restraint gross misuse were excluded. Occupants with preexisting comorbidities contributing to spinal injury and occupants with limited injury information were also excluded. A multidisciplinary team retrospectively reviewed each case to determine injury causation scenarios (ICSs). Crash conditions, occupant and restraint characteristics, and injuries were qualitatively summarized. Results Fifty-nine cases met the study inclusion criteria and 17 were excluded. The 42 occupants included sustained 97 distinct AIS 2+ spinal injuries (27 cervical, 22 thoracic, and 48 lumbar; 80 AIS-2, 15 AIS-3, 1 AIS-5, and 1 AIS-6), with fracture as the most common injury type (80%). Spinal-injured occupants were most frequently in passenger cars (64%), and crash direction was most often frontal (62%). Mean delta-V was 51.3 km/h ± 19.4 km/h. The average occupant age was 12.4 ± 5.3 years old, and 48% were 16- to 17-year-olds. Thirty-six percent were right front passengers and 26% were drivers. Most occupants were lap and shoulder belt restrained (88%). Non-spinal AIS 2+ injuries included those of the lower extremity and pelvis (n = 56), head (n = 43), abdomen (n = 39), and thorax (n = 36). Spinal injury causation was typically due to flexion or lateral bending over the lap and or shoulder belt or child restraint harness, compression by occupant�

Efficacy of tranexamic acid plus drain-clamping to reduce blood loss in total knee arthroplasty

PubMed Central

Zhang, Yan; Zhang, Jun-Wei; Wang, Bao-Hua

2017-01-01

Abstract Background: Perioperative blood loss is still an unsolved problem in total knee arthroplasty (TKA). The efficacy of the preoperative use of tranexamic acid (TXA) plus drain-clamping to reduce blood loss in TKA has been debated. This meta-analysis aimed to illustrate the efficacy of TXA plus drain-clamping to reduce blood loss in patients who underwent a TKA. Methods: In February 2017, a systematic computer-based search was conducted in PubMed, EMBASE, Web of Science, the Cochrane Database of Systematic Reviews, and Google Scholar. Data from patients prepared for TKA in studies that compared TXA plus drain-clamping versus TXA alone, drain-clamping alone, or controls were retrieved. The primary endpoint was the need for transfusion. The secondary outcomes were total blood loss, blood loss in drainage, the decrease in hemoglobin, and the occurrence of deep venous thrombosis. After testing for publication bias and heterogeneity between studies, data were aggregated for random-effects models when necessary. Results: Ultimately, 5 clinical studies with 618 patients (TXA plus drain-clamping group = 249, control group = 130, TXA-alone group = 60, and drain-clamping group = 179) were included. TXA plus drain-clamping could decrease the need for transfusion, total blood loss, blood loss in drainage, and the decrease in hemoglobin than could the control group, the TXA-alone group, and the drain-clamping group (P < .05). There was no significant difference between the occurrence of deep venous thrombosis between the included groups (P > .05). Conclusions: TXA plus drain-clamping can achieve the maximum effects of hemostasis in patients prepared for primary TKA. Because the number and the quality of the included studies were limited, more high-quality randomized controlled trials are needed to identify the optimal dose of TXA and the clamping hours in patients prepared for TKA. PMID:28658157

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

PubMed Central

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

RNA polymerase pausing and nascent RNA structure formation are linked through clamp domain movement

PubMed Central

Hein, Pyae P.; Kolb, Kellie E.; Windgassen, Tricia; Bellecourt, Michael J.; Darst, Seth A.; Mooney, Rachel A.; Landick, Robert

2014-01-01

The rates of RNA synthesis and nascent RNA folding into biologically active structures are linked via pausing by RNA polymerase (RNAP). Structures that form within the RNA exit channel can increase pausing by interacting with bacterial RNAP or decrease pausing by preventing backtracking. Conversely, pausing is required for proper folding of some RNAs. Opening of the RNAP clamp domain is proposed to mediate some effects of nascent RNA structures. However, the connections among RNA structure formation, clamp movement, and catalytic activity remain uncertain. We assayed exit-channel structure formation in Escherichia coli RNAP together with disulfide crosslinks that favor closed or open clamp conformations and found that clamp position directly influences RNA structure formation and catalytic activity. We report that exit-channel RNA structures slow pause escape by favoring clamp opening and through interactions with the flap that slow translocation. PMID:25108353

76 FR 22120 - Credit Watch Termination Initiative; Termination of Origination Approval Agreements

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-20

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Midwives in India: a delayed cord clamping intervention using simulation.

PubMed

Faucher, M A; Riley, C; Prater, L; Reddy, M P

2016-09-01

Iron deficiency is a prevalent health problem in India affecting women and newborns. Delayed umbilical cord clamping at birth is a safe and effective means for increasing serum iron levels in newborns up to 6 months of age. The study aim was to increase the utilization of delayed cord clamping in a group of midwives working in Hyderabad, India. A single group pre- and post-test design was used to evaluate knowledge, beliefs and practice before and after a delayed cord clamping intervention including follow-up at 10 months after the original intervention. The intervention included lectures and simulation. Results show significant increases in knowledge and positive beliefs about the practice of delayed cord clamping. Simulation was effective for eliciting important feedback related to learning. Results represent a small group of midwives working with a non-profit foundation in Southern India. Language discordancy and cultural norms in this group of midwives may have influenced results. Knowledge, beliefs and practice related to delayed cord clamping were all significantly improved after the intervention. The Knowledge to Action framework using simulation is an effective cross-cultural method for implementing education about evidence-based practice. Midwives are invested in learning practices that promote public health. Changing institutional policy may have limitations without first considering normative practice. Using simulation combined with institutional health policy appears to result in significant uptake of practice change. Qualitative studies exploring the interconnections between cultural norms and decision making may be informative about promoting practice change particularly in this setting. Upscaling midwifery has been recommended to improve maternal and child health in India. © 2016 International Council of Nurses.

Conformation changes, N-terminal involvement and cGMP signal relay in phosphodiesterase-5 GAF domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, H.; Robinson, H.; Ke, H.

2010-12-03

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

C-Terminal residues in small potassium channel blockers OdK1 and OSK3 from scorpion venom fine-tune the selectivity.

PubMed

Kuzmenkov, Alexey I; Peigneur, Steve; Chugunov, Anton O; Tabakmakher, Valentin M; Efremov, Roman G; Tytgat, Jan; Grishin, Eugene V; Vassilevski, Alexander A

2017-05-01

We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC 50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity. Copyright © 2017 Elsevier B.V. All rights reserved.

QPatch: the past, present and future of automated patch clamp.

PubMed

Mathes, Chris

2006-04-01

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

High Efficiency Automatic-Power-Controlled and Gain-Clamped EDFA for Broadband Passive Optical Networking Systems

NASA Astrophysics Data System (ADS)

Shen, Jyi-Lai; Wei, Shui-Ken; Lin, Chin-Yuan; Iong Li, Ssu; Huang, Chih-Chuan

2010-04-01

The configuration of a simple improved high efficiency automatic-power-controlled and gain-clamped EDFA (APC-GC-EDFA) for broadband passive optical networking systems (BPON) is presented here. In order to compensate the phase and amplitude variation due to the different distance between the optical line terminal (OLT) and optical network units (ONU), the APC-GC-EDFA need to be employed. A single 980 nm laser module is employed as the primary pump. To extend the bandwidth, all C-band ASE is recycled as the secondary pump to enhance the gain efficiency. An electrical feedback circuit is used as a multi-wavelength channel transmitter monitor for the automatic power control to improve the gain-flattened flatness for stable amplification. The experimental results prove that the EDFA system can provide flatter clamped gain in both C-band and L-band configurations. The gain flatness wavelength ranging from 1530 to 1610 nm is within 32.83 ± 0.64 dB, i.e. below 1.95 %. The gains are clamped at 33.85 ± 0.65 dB for the input signal power of -40 dBm to -10 dBm. The range of noise figure is between 6.37 and 6.56, which is slightly lower compared to that of unclamped amplifiers. This will be very useful for measuring the gain flatness of APC-GC-EDFA. Finally, we have also demonstrated the records of the overall simultaneous dynamics measurements for the new system stabilization. The carrier to noise ratio (CNR) is 49.5 to 50.8 dBc which is above the National Television System Committee (NTSC) standard of 43 dBc, and both composite second order (CSO) 69.2 to 71.5 dBc and composite triple beat (CTB) of 69.8 to 72.2 dBc are above 53 dBc. The recorded corresponding rise-time of 1.087 ms indicates that the system does not exhibit any overshoot of gain or ASE variation due to the signal at the beginning of the pulse.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

PubMed

Kim, Jin S; Nanfara, Michael T; Chodavarapu, Sundari; Jin, Kyeong S; Babu, Vignesh M P; Ghazy, Mohamed A; Chung, Scisung; Kaguni, Jon M; Sutton, Mark D; Cho, Yunje

2017-04-20

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.

PubMed

Abbott, Geoffrey W

2016-08-01

The 5 human (h)KCNE β subunits each regulate various cation channels and are linked to inherited cardiac arrhythmias. Reported here are previously undiscovered protein-coding regions in exon 1 of hKCNE3 and hKCNE4 that extend their encoded extracellular domains by 44 and 51 residues, which yields full-length proteins of 147 and 221 residues, respectively. Full-length hKCNE3 and hKCNE4 transcript and protein are expressed in multiple human tissues; for hKCNE4, only the longer protein isoform is detectable. Two-electrode voltage-clamp electrophysiology revealed that, when coexpressed in Xenopus laevis oocytes with various potassium channels, the newly discovered segment preserved conversion of KCNQ1 by hKCNE3 to a constitutively open channel, but prevented its inhibition of Kv4.2 and KCNQ4. hKCNE4 slowing of Kv4.2 inactivation and positive-shifted steady-state inactivation were also preserved in the longer form. In contrast, full-length hKCNE4 inhibition of KCNQ1 was limited to 40% at +40 mV vs. 80% inhibition by the shorter form, and augmentation of KCNQ4 activity by hKCNE4 was entirely abolished by the additional segment. Among the genome databases analyzed, the longer KCNE3 is confined to primates; full-length KCNE4 is widespread in vertebrates but is notably absent from Mus musculus Findings highlight unexpected KCNE gene diversity, raise the possibility of dynamic regulation of KCNE partner modulation via splice variation, and suggest that the longer hKCNE3 and hKCNE4 proteins should be adopted in future mechanistic and genetic screening studies.-Abbott, G. W. Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4. © FASEB.

Automatic Approach Tendencies toward High and Low Caloric Food in Restrained Eaters: Influence of Task-Relevance and Mood

PubMed Central

Neimeijer, Renate A. M.; Roefs, Anne; Ostafin, Brian D.; de Jong, Peter J.

2017-01-01

Objective: Although restrained eaters are motivated to control their weight by dieting, they are often unsuccessful in these attempts. Dual process models emphasize the importance of differentiating between controlled and automatic tendencies to approach food. This study investigated the hypothesis that heightened automatic approach tendencies in restrained eaters would be especially prominent in contexts where food is irrelevant for their current tasks. Additionally, we examined the influence of mood on the automatic tendency to approach food as a function of dietary restraint. Methods: An Affective Simon Task-manikin was administered to measure automatic approach tendencies where food is task-irrelevant, and a Stimulus Response Compatibility task (SRC) to measure automatic approach in contexts where food is task-relevant, in 92 female participants varying in dietary restraint. Prior to the task, sad, stressed, neutral, or positive mood was induced. Food intake was measured during a bogus taste task after the computer tasks. Results: Consistent with their diet goals, participants with a strong tendency to restrain their food intake showed a relatively weak approach bias toward food when food was task-relevant (SRC) and this effect was independent of mood. Restrained eaters showed a relatively strong approach bias toward food when food was task-irrelevant in the positive condition and a relatively weak approach in the sad mood. Conclusion: The weak approach bias in contexts where food is task-relevant may help high-restrained eaters to comply with their diet goal. However, the strong approach bias in contexts where food is task-irrelevant and when being in a positive mood may interfere with restrained eaters’ goal of restricting food-intake. PMID:28443045

Whole-Cell Electrical Activity Under Direct Mechanical Stimulus by AFM Cantilever Using Planar Patch Clamp Chip Approach

PubMed Central

Upadhye, Kalpesh V.; Candiello, Joseph E.; Davidson, Lance A.; Lin, Hai

2011-01-01

Patch clamp is a powerful tool for studying the properties of ion-channels and cellular membrane. In recent years, planar patch clamp chips have been fabricated from various materials including glass, quartz, silicon, silicon nitride, polydimethyl-siloxane (PDMS), and silicon dioxide. Planar patch clamps have made automation of patch clamp recordings possible. However, most planar patch clamp chips have limitations when used in combination with other techniques. Furthermore, the fabrication methods used are often expensive and require specialized equipments. An improved design as well as fabrication and characterization of a silicon-based planar patch clamp chip are described in this report. Fabrication involves true batch fabrication processes that can be performed in most common microfabrication facilities using well established MEMS techniques. Our planar patch clamp chips can form giga-ohm seals with the cell plasma membrane with success rate comparable to existing patch clamp techniques. The chip permits whole-cell voltage clamp recordings on variety of cell types including Chinese Hamster Ovary (CHO) cells and pheochromocytoma (PC12) cells, for times longer than most available patch clamp chips. When combined with a custom microfluidics chamber, we demonstrate that it is possible to perfuse the extra-cellular as well as intra-cellular buffers. The chamber design allows integration of planar patch clamp with atomic force microscope (AFM). Using our planar patch clamp chip and microfluidics chamber, we have recorded whole-cell mechanosensitive (MS) currents produced by directly stimulating human keratinocyte (HaCaT) cells using an AFM cantilever. Our results reveal the spatial distribution of MS ion channels and temporal details of the responses from MS channels. The results show that planar patch clamp chips have great potential for multi-parametric high throughput studies of ion channel proteins. PMID:22174731

Acoustic plane waves incident on an oblique clamped panel in a rectangular duct

NASA Technical Reports Server (NTRS)

Unz, H.; Roskam, J.

1980-01-01

The theory of acoustic plane waves incident on an oblique clamped panel in a rectangular duct was developed from basic theoretical concepts. The coupling theory between the elastic vibrations of the panel (plate) and the oblique incident acoustic plane wave in infinite space was considered in detail, and was used for the oblique clamped panel in the rectangular duct. The partial differential equation which governs the vibrations of the clamped panel (plate) was modified by adding to it stiffness (spring) forces and damping forces. The Transmission Loss coefficient and the Noise Reduction coefficient for oblique incidence were defined and derived in detail. The resonance frequencies excited by the free vibrations of the oblique finite clamped panel (plate) were derived and calculated in detail for the present case.

[Modulation of Kv4 channels by KChIPs clamping].

PubMed

Cui, Yuan-Yuan; Wang, Ke-Wei

2009-01-01

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs co-assemble with Kv4 (Shal) alpha subunits to form a native complex. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Based on recent structural efforts, here we attempt to emphasize the interaction between KChIPs and Kv4 channel complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials by structure-based design of chemical compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

Zonal variations in K+ currents in vestibular crista calyx terminals

PubMed Central

Meredith, Frances L.

2014-01-01

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na+ but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K+ currents were observed at potentials above −60 mV. K+ currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K+ channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K+ channel blocker 4-aminopyridine (10–50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K+ conductances may contribute to different firing responses in calyx afferents. PMID:25343781

Zonal variations in K+ currents in vestibular crista calyx terminals.

PubMed

Meredith, Frances L; Rennie, Katherine J

2015-01-01

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na(+) but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K(+) currents were observed at potentials above -60 mV. K(+) currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K(+) channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K(+) channel blocker 4-aminopyridine (10-50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K(+) conductances may contribute to different firing responses in calyx afferents. Copyright © 2015 the American Physiological Society.

Timing of cord clamping in very preterm infants: more evidence is needed.

PubMed

Tarnow-Mordi, William O; Duley, Lelia; Field, David; Marlow, Neil; Morris, Jonathan; Newnham, John; Paneth, Nigel; Soll, Roger F; Sweet, David

2014-08-01

In December 2012, the American College of Obstetricians and Gynecologists published a Committee Opinion entitled "Timing of umbilical cord clamping after birth." It stated that "evidence exists to support delayed cord clamping in preterm infants, when feasible. The single most important benefit for preterm infants is the possibility for a nearly 50% reduction in IVH." However, the Committee Opinion added that the ideal timing of umbilical cord clamping has yet to be determined and recommended that large clinical trials be conducted in the most preterm infants. Published randomized controlled trials include <200 infants of <30 weeks' gestation, with assessments of neurodevelopmental outcome in less than one-half of the children. This is a major gap in the evidence. Without reliable data from randomized controlled trials that optimally include childhood follow-up evaluations, we will not know whether delayed cord clamping may do more overall harm than good. Ongoing trials of delayed cord clamping plan to report childhood outcomes in >2000 additional very preterm infants. Current recommendations may need to change when these results become available. Greater international collaboration could accelerate resolution of whether this promising intervention will improve disability-free survival in about 1 million infants who will be born very preterm globally each year. Copyright © 2014 Mosby, Inc. All rights reserved.

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

Measuring true Young's modulus of a cantilevered nanowire: effect of clamping on resonance frequency.

PubMed

Qin, Qingquan; Xu, Feng; Cao, Yongqing; Ro, Paul I; Zhu, Yong

2012-08-20

The effect of clamping on resonance frequency and thus measured Young's modulus of nanowires (NWs) is systematically investigated via a combined experimental and simulation approach. ZnO NWs are used in this work as an example. The resonance tests are performed in situ inside a scanning electron microscope and the NWs are cantilevered on a tungsten probe by electron-beam-induced deposition (EBID) of hydrocarbon. EBID is repeated several times to deposit more hydrocarbons at the same location. The resonance frequency increases with the increasing clamp size until approaching that under the "fixed" boundary condition. The critical clamp size is identified as a function of NW diameter and NW Young's modulus. This work: 1) exemplifies the importance of considering the effect of clamping in measurements of Young's modulus using the resonance method, and 2) demonstrates that the true Young's modulus can be measured if the critical clamp size is reached. Design guidelines on the critical clamp size are provided. Such design guidelines can be extended to other one-dimensional nanostructures such as carbon nanotubes. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Termination unit

DOEpatents

Traeholt, Chresten; Willen, Dag; Roden, Mark; Tolbert, Jerry C.; Lindsay, David; Fisher, Paul W.; Nielsen, Carsten Thidemann

2016-05-03

Cable end section comprises end-parts of N electrical phases/neutral, and a thermally-insulation envelope comprising cooling fluid. The end-parts each comprises a conductor and are arranged with phase 1 innermost, N outermost surrounded by the neutral, electrical insulation being between phases and N and neutral. The end-parts comprise contacting surfaces located sequentially along the longitudinal extension of the end-section. A termination unit has an insulating envelope connected to a cryostat, special parts at both ends comprising an adapter piece at the cable interface and a closing end-piece terminating the envelope in the end-section. The special parts houses an inlet and/or outlet for cooling fluid. The space between an inner wall of the envelope and a central opening of the cable is filled with cooling fluid. The special part at the end connecting to the cryostat houses an inlet or outlet, splitting cooling flow into cable annular flow and termination annular flow.

A miniaturized planar patch-clamp system for transportable use.

PubMed

Boussaoud, Adrien; Fonteille, Isabelle; Collier, Guilhem; Kermarrec, Frédérique; Vermont, Fabien; Tresallet, Eric; De Waard, Michel; Arnoult, Christophe; Picollet-D'hahan, Nathalie

2012-02-15

In the last decade, planar patch-clamp (PPC) has emerged as an innovative technology allowing parallel recordings of cellular electrophysiological activity on planar substrates. If PPC is widely adopted by the pharmaceutical sector, it remains poorly extended to other areas (i.e. environment and safety organizations) probably because of the large, expensive and non-easily transportable format of those commercial equipments. The present work describes for the first time a new compact and transportable planar patch-clamp system (named Toxint'patch or TIP, for Toxin detection with integrated patch-clamp) focusing on environmental matters and meant to be used in coastal laboratories, for direct on-site monitoring of the seawater and shellfish quality. The TIP system incorporates silicon chips tailored to monitor cellular ionic currents from cultured cells stably expressing a phycotoxin molecular target. The functionality of this novel briefcase-sized PPC system is described in terms of fluidic control, electronic performances with amplifying and filtering boards and of user interface for data acquisition and control implemented on a computer. Copyright © 2011 Elsevier B.V. All rights reserved.

Vertical GaN power diodes with a bilayer edge termination

DOE PAGES

Dickerson, Jeramy R.; Allerman, Andrew A.; Bryant, Benjamin N.; ...

2015-12-07

Vertical GaN power diodes with a bilayer edge termination (ET) are demonstrated. The GaN p-n junction is formed on a low threading dislocation defect density (10 4 - 10 5 cm -2) GaN substrate, and has a 15-μm-thick n-type drift layer with a free carrier concentration of 5 × 10 15 cm -3. The ET structure is formed by N implantation into the p+-GaN epilayer just outside the p-type contact to create compensating defects. The implant defect profile may be approximated by a bilayer structure consisting of a fully compensated layer near the surface, followed by a 90% compensated (p)more » layer near the n-type drift region. These devices exhibit avalanche breakdown as high as 2.6 kV at room temperature. In addition simulations show that the ET created by implantation is an effective way to laterally distribute the electric field over a large area. This increases the voltage at which impact ionization occurs and leads to the observed higher breakdown voltages.« less

A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

PubMed Central

Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

2012-01-01

The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

The unique C- and N-terminal sequences of Metallothionein isoform 3 mediate growth inhibition and Vectorial active transport in MCF-7 cells.

PubMed

Voels, Brent; Wang, Liping; Sens, Donald A; Garrett, Scott H; Zhang, Ke; Somji, Seema

2017-05-25

The 3rd isoform of the metallothionein (MT3) gene family has been shown to be overexpressed in most ductal breast cancers. A previous study has shown that the stable transfection of MCF-7 cells with the MT3 gene inhibits cell growth. The goal of the present study was to determine the role of the unique C-terminal and N-terminal sequences of MT3 on phenotypic properties and gene expression profiles of MCF-7 cells. MCF-7 cells were transfected with various metallothionein gene constructs which contain the insertion or the removal of the unique MT3 C- and N-terminal domains. Global gene expression analysis was performed on the MCF-7 cells containing the various constructs and the expression of the unique C- and N- terminal domains of MT3 was correlated to phenotypic properties of the cells. The results of the present study demonstrate that the C-terminal sequence of MT3, in the absence of the N-terminal sequence, induces dome formation in MCF-7 cells, which in cell cultures is the phenotypic manifestation of a cell's ability to perform vectorial active transport. Global gene expression analysis demonstrated that the increased expression of the GAGE gene family correlated with dome formation. Expression of the C-terminal domain induced GAGE gene expression, whereas the N-terminal domain inhibited GAGE gene expression and that the effect of the N-terminal domain inhibition was dominant over the C-terminal domain of MT3. Transfection with the metallothionein 1E gene increased the expression of GAGE genes. In addition, both the C- and the N-terminal sequences of the MT3 gene had growth inhibitory properties, which correlated to an increased expression of the interferon alpha-inducible protein 6. Our study shows that the C-terminal domain of MT3 confers dome formation in MCF-7 cells and the presence of this domain induces expression of the GAGE family of genes. The differential effects of MT3 and metallothionein 1E on the expression of GAGE genes suggests unique roles of

Strategy of restraining ripple error on surface for optical fabrication.

PubMed

Wang, Tan; Cheng, Haobo; Feng, Yunpeng; Tam, Honyuen

2014-09-10

The influence from the ripple error to the high imaging quality is effectively reduced by restraining the ripple height. A method based on the process parameters and the surface error distribution is designed to suppress the ripple height in this paper. The generating mechanism of the ripple error is analyzed by polishing theory with uniform removal character. The relation between the processing parameters (removal functions, pitch of path, and dwell time) and the ripple error is discussed through simulations. With these, the strategy for diminishing the error is presented. A final process is designed and demonstrated on K9 work-pieces using the optimizing strategy with magnetorheological jet polishing. The form error on the surface is decreased from 0.216λ PV (λ=632.8  nm) and 0.039λ RMS to 0.03λ PV and 0.004λ RMS. And the ripple error is restrained well at the same time, because the ripple height is less than 6 nm on the final surface. Results indicate that these strategies are suitable for high-precision optical manufacturing.

Effect of sodium chloride on the structure and stability of spider silk's N-terminal protein domain.

PubMed

Gronau, Greta; Qin, Zhao; Buehler, Markus J

2013-03-01

A spider's ability to store silk protein solutions at high concentration is believed to be related to the protein's terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage.

[Advantage of delayed umbilical cord clamping in the newborn infant].

PubMed

Menget, A; Mougey, C; Thiriez, G; Riethmuller, D

2013-09-01

The timing of umbilical cord clamping remains controversial. Although most maternity wards use the early clamping (5-15s), randomized studies and meta-analyses have demonstrated the benefit of delayed clamping for term and preterm newborn infants over the past 10 years. Indeed, placentofetal transfusion of 20-30 ml/kg in 2-3 min improves the iron status of term infants and prevents infant hypochromic anemia. Infant anemia is a public health problem in many developing countries. For preterm newborns, placental transfusion for 45 s or milking the cord for 15 s improves cardiovascular adaptation, with better hemodynamic stability, as well as decreased intraventricular hemorrhages, need for transfusion, and late-onset sepsis. A new look at this symbolic act is needed and professionals need to be persuaded of the importance of the "wait a minute" policy for a better physiological delivery. Copyright © 2013. Published by Elsevier SAS.

Responsiveness to healthy advertisements in adults: An experiment assessing beyond brand snack selection and the impact of restrained eating.

PubMed

Dovey, Terence M; Torab, Tina; Yen, Dorothy; Boyland, E J; Halford, Jason C G

2017-05-01

The objective of this study was to explore the impact of different advertising messages on adults' snack choice. Eighty participants (18-24 years old) were offered the choice between two snack packs following exposure to one of three advertising conditions. The snack packs contained either healthy or high fat, sugar or salt (HFSS) foods. Participants were exposed to commercials containing either non-food products, healthy food products or HFSS food products and their subsequent choice of snack pack was recorded. The Dutch Eating Behaviour Questionnaire (DEBQ) was used to assess the impact of external, restrained and emotional eating behaviour on snack pack selection following exposure to advertisements. The majority of unrestrained participants preferentially choose the HFSS snack pack irrespective of advertisement condition. In contrast, high restrained individuals exposed to the healthy eating advertisement condition preferentially selected the healthy snack pack while those in other advertisement conditions refused to take either snack pack. The healthy eating message, when distributed through mass media, resonated with restrained eaters only. Exposure to healthy food adverts provoked restrained eaters into choosing a snack pack; while exposure to other messages results in restrained eaters refusing to take any foods. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Characterization of GABAA receptor ligands with automated patch-clamp using human neurons derived from pluripotent stem cells

PubMed Central

Yuan, Nina Y.; Poe, Michael M.; Witzigmann, Christopher; Cook, James M.; Stafford, Douglas; Arnold, Leggy A.

2016-01-01

Introduction Automated patch clamp is a recent but widely used technology to assess pre-clinical drug safety. With the availability of human neurons derived from pluripotent stem cells, this technology can be extended to determine CNS effects of drug candidates, especially those acting on the GABAA receptor. Methods iCell Neurons (Cellular Dynamics International, A Fujifilm Company) were cultured for ten days and analyzed by patch clamp in the presence of agonist GABA or in combination with positive allosteric GABAA receptor modulators. Both efficacy and affinity were determined. In addition, mRNA of GABAA receptor subunits were quantified by qRT-PCR. Results We have shown that iCell Neurons are compatible with the IonFlux microfluidic system of the automated patch clamp instrument. Resistance ranging from 15-25 MΩ was achieved for each trap channel of patch clamped cells in a 96-well plate format. GABA induced a robust change of current with an EC50 of 0.43 μM. Positive GABAA receptor modulators diazepam, HZ166, and CW-04-020 exhibited EC50 values of 0.42 μM, 1.56 μM, and 0.23 μM, respectively. The α2/α3/α5 selective compound HZ166-induced the highest potentiation (efficacy) of 810% of the current induced by 100 nM GABA. Quantification of GABAA receptor mRNA in iCell Neurons revealed high levels of α5 and β3 subunits and low levels of α1, which is similar to the configuration in human neonatal brain. Discussion iCell Neurons represent a new cellular model to characterize GABAergic compounds using automated patch clamp. These cells have excellent representation of cellular GABAA receptor distribution that enable determination of total small molecule efficacy and affinity as measured by cell membrane current change. PMID:27544543

A nudge in a healthier direction: How environmental cues help restrained eaters pursue their weight-control goal.

PubMed

Stämpfli, Aline E; Stöckli, Sabrina; Brunner, Thomas A

2017-03-01

Losing weight is a goal for many people, but it is hard to pursue. However, dieting cues in the environment hold promise for improving individuals' eating behavior. For example, exposure to thin, human-like sculptures by the artist Alberto Giacometti has been found to promote healthy snack choices at a vending machine. Whether health- or weight-related processes drive such effects has not yet been determined. However, a detailed understanding of the content-related drivers of environmental cues' effects provides the first indications regarding a cue's possible use. Therefore, two laboratory studies were conducted. They examined the Giacometti sculptures' effects on unhealthy and healthy food intake (Study 1) and on the completion of weight- and health-related fragmented words (Study 2). Study 1 indicated that the sculptures are weight-related by showing that they reduced food intake independent of food healthiness. Furthermore, the "Giacometti effect" was moderated by restrained eating. Restrained eaters, who are known for their weight-control goal, ate less after having been exposed to the thin sculptures. The results of Study 2 pointed in the same direction. Restrained eaters completed more weight-related words after being exposed to the sculptures. Overall, these studies suggest that the thin sculptures are primarily weight-related cues and particularly helpful for restrained eaters. Environmental weight-control cues such as the Giacometti sculptures could act as a counterforce to our obesogenic environment and help restrained eaters pursue their weight-control goal. In this way, they could nudge food decisions in a healthier direction. Copyright © 2016 Elsevier Ltd. All rights reserved.

HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor.

PubMed

Chain, Benjamin M; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward

2008-10-23

The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.

HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor

PubMed Central

Chain, Benjamin M.; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward

2008-01-01

The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes. PMID:18765264

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

PubMed

Kawakami, Hironori; Su'etsugu, Masayuki; Katayama, Tsutomu

2006-10-01

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

The unique N-terminal zinc finger of synaptotagmin-like protein 4 reveals FYVE structure.

PubMed

Miyamoto, Kazuhide; Nakatani, Arisa; Saito, Kazuki

2017-12-01

Synaptotagmin-like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N-terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N-terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross-brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C 4 C 4 -type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin-conjugating enzyme (E2)-binding capability, cross-brace structures with eight zinc-ligating residues are needed as the scaffold. The cross-brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs. © 2017 The Protein Society.

76 FR 22119 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

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2011-04-20

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76 FR 53148 - Credit Watch Termination Initiative; Termination of Direct Endorsement (DE) Approval

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77 FR 5262 - Credit Watch Termination Initiative Termination of Direct Endorsement (DE) Approval

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Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

PubMed Central

Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

2014-01-01

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

A novel monolithic piezoelectric actuated flexure-mechanism based wire clamp for microelectronic device packaging.

PubMed

Liang, Cunman; Wang, Fujun; Tian, Yanling; Zhao, Xingyu; Zhang, Hongjie; Cui, Liangyu; Zhang, Dawei; Ferreira, Placid

2015-04-01

A novel monolithic piezoelectric actuated wire clamp is presented in this paper to achieve fast, accurate, and robust microelectronic device packaging. The wire clamp has compact, flexure-based mechanical structure and light weight. To obtain large and robust jaw displacements and ensure parallel jaw grasping, a two-stage amplification composed of a homothetic bridge type mechanism and a parallelogram leverage mechanism was designed. Pseudo-rigid-body model and Lagrange approaches were employed to conduct the kinematic, static, and dynamic modeling of the wire clamp and optimization design was carried out. The displacement amplification ratio, maximum allowable stress, and natural frequency were calculated. Finite element analysis (FEA) was conducted to evaluate the characteristics of the wire clamp and wire electro discharge machining technique was utilized to fabricate the monolithic structure. Experimental tests were carried out to investigate the performance and the experimental results match well with the theoretical calculation and FEA. The amplification ratio of the clamp is 20.96 and the working mode frequency is 895 Hz. Step response test shows that the wire clamp has fast response and high accuracy and the motion resolution is 0.2 μm. High speed precision grasping operations of gold and copper wires were realized using the wire clamper.

N-Terminal Domain of Turkey Pancreatic Lipase is Active on Long Chain Triacylglycerols and Stabilized by Colipase

PubMed Central

Bou Ali, Madiha; Karray, Aida; Gargouri, Youssef; Ben Ali, Yassine

2013-01-01

The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin–water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain. PMID:23977086

Acute phase response in lactating dairy cows during hyperinsulinemic hypoglycaemic and hyperinsulinemic euglycaemic clamps and after intramammary LPS challenge.

PubMed

De Matteis, L; Bertoni, G; Lombardelli, R; Wellnitz, O; Van Dorland, H A; Vernay, M C M B; Bruckmaier, R M; Trevisi, E

2017-06-01

The link between energy availability, turnover of energy substrates and the onset of inflammation in dairy cows is complex and poorly investigated. To clarify this, plasma inflammatory variables were measured in mid-lactating dairy cows allocated to three groups: hyperinsulinemic hypoglycaemic clamp, induced by insulin infusion (HypoG, n = 5); hyperinsulinemic euglycaemic clamp, induced by insulin and glucose infusion (EuG; n = 6); control, receiving a saline solution infusion (NaCl; n = 6). At 48 h after the start of i.v. infusions, two udder quarters per cow were challenged with 200 μg of E. coli lipopolysaccharide (LPS). Individual blood samples were taken before clamps, before LPS challenge (i.e. 48 h after clamps) and 6.5 h after. At 48 h, positive acute phase proteins (posAPP) did not differ among groups, whereas albumin and cholesterol (index of lipoproteins), negative APP (negAPP), were lower (p < 0.05) in EuG compared to NaCl and HypoG. The concentration of IL-6 was greater in EuG (p < 0.05) but only vs. HypoG. At 6.5 h following LPS challenge, IL-6 increased in the NaCl and EuG clamps (p < 0.05), while TNF-α increased (p < 0.05) in the EuG only. Among the posAPP, haptoglobin markedly increased in EuG (p < 0.05), but not in NaCl (p = 0.76) and in HypoG; ceruloplasmin tended to decline during LPS challenge, the reduction was significant when all animals were considered (p < 0.05). Conversely, all the negAPP showed a marked reduction 6.5 h after LPS challenge in the three groups. In conclusion, EuG caused an inflammatory status after 48-h infusion (i.e. decrease of negAPP) and induced a quicker acute phase response (e.g. marked rise of TNF-α, IL-6) after the intramammary LPS challenge. These data suggest that the simultaneous high availability of glucose and insulin at the tissue-level makes dairy cows more susceptible to inflammatory events. In contrast, HypoG seems to attenuate the inflammatory response. Journal of Animal

HTS techniques for patch clamp-based ion channel screening - advances and economy.

PubMed

Farre, Cecilia; Fertig, Niels

2012-06-01

Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. "Going planar" proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Non-native, N-terminal Hsp70 Molecular Motor Recognition Elements in Transit Peptides Support Plastid Protein Translocation*

PubMed Central

Chotewutmontri, Prakitchai; Bruce, Barry D.

2015-01-01

Previously, we identified the N-terminal domain of transit peptides (TPs) as a major determinant for the translocation step in plastid protein import. Analysis of Arabidopsis TP dataset revealed that this domain has two overlapping characteristics, highly uncharged and Hsp70-interacting. To investigate these two properties, we replaced the N-terminal domains of the TP of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and its reverse peptide with a series of unrelated peptides whose affinities to the chloroplast stromal Hsp70 have been determined. Bioinformatic analysis indicated that eight out of nine peptides in this series are not similar to the TP N terminus. Using in vivo and in vitro protein import assays, the majority of the precursors containing Hsp70-binding elements were targeted to plastids, whereas none of the chimeric precursors lacking an N-terminal Hsp70-binding element were targeted to the plastids. Moreover, a pulse-chase assay showed that two chimeric precursors with the most uncharged peptides failed to translocate into the stroma. The ability of multiple unrelated Hsp70-binding elements to support protein import verified that the majority of TPs utilize an N-terminal Hsp70-binding domain during translocation and expand the mechanistic view of the import process. This work also indicates that synthetic biology may be utilized to create de novo TPs that exceed the targeting activity of naturally occurring sequences. PMID:25645915

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier

2014-01-03

Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence uniquemore » in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.« less

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

PubMed

Nguyen, Khoa; Garcia, Alvaro; Sani, Marc-Antoine; Diaz, Dil; Dubey, Vikas; Clayton, Daniel; Dal Poggetto, Giovanni; Cornelius, Flemming; Payne, Richard J; Separovic, Frances; Khandelia, Himanshu; Clarke, Ronald J

2018-06-01

The Na + ,K + -ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na + and K + electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31 P and 2 H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations. Copyright © 2018 Elsevier B.V. All rights reserved.

Preparation of protein samples for mass spectrometry and N-terminal sequencing.

PubMed

Glenn, Gary

2014-01-01

The preparation of protein samples for mass spectrometry and N-terminal sequencing is a key step in successfully identifying proteins. Mass spectrometry is a very sensitive technique, and as such, samples must be prepared carefully since they can be subject to contamination of the sample (e.g., due to incomplete subcellular fractionation or purification of a multiprotein complex), overwhelming of the sample by highly abundant proteins, and contamination from skin or hair (keratin can be a very common hit). One goal of sample preparation for mass spec is to reduce the complexity of the sample - in the example presented here, mitochondria are purified, solubilized, and fractionated by sucrose density gradient sedimentation prior to preparative 1D SDS-PAGE. It is important to verify the purity and integrity of the sample so that you can have confidence in the hits obtained. More protein is needed for N-terminal sequencing and ideally it should be purified to a single band when run on an SDS-polyacrylamide gel. The example presented here involves stably expressing a tagged protein in HEK293 cells and then isolating the protein by affinity purification and SDS-PAGE. © 2014 Elsevier Inc. All rights reserved.

An N-terminal glycine-rich sequence contributes to retrovirus trimer of hairpins stability

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilson, Kirilee A.; Maerz, Anne L.; Baer, Severine

2007-08-10

Retroviral transmembrane proteins (TMs) contain a glycine-rich segment linking the N-terminal fusion peptide and coiled coil core. Previously, we reported that the glycine-rich segment (Met-326-Ser-337) of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, is a determinant of membrane fusion function [K.A. Wilson, S. Baer, A.L. Maerz, M. Alizon, P. Poumbourios, The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function, J. Virol. 79 (2005) 4533-4539]. Here we show that the reduced fusion activity of an I334A mutantmore » correlated with a decrease in stability of the gp21 trimer of hairpins conformation, in the context of a maltose-binding protein-gp21 chimera. The stabilizing influence of Ile-334 required the C-terminal membrane-proximal sequence Trp-431-Ser-436. Proline substitution of four of five Gly residues altered gp21 trimer of hairpins stability. Our data indicate that flexibility within and hydrophobic interactions mediated by this region are determinants of gp21 stability and membrane fusion function.« less

Tensile film clamps and mounting block for the rheovibron and autovibron viscoelastometer

NASA Technical Reports Server (NTRS)

Stoakley, Diane M. (Inventor); St.clair, Anne K. (Inventor); Little, Bruce D. (Inventor)

1989-01-01

A set of film clamps and a mounting block for use in the determination of tensile modulus and damping properties of films in a manually operated or automated Rheovibron is diagrammed. These clamps and mounting block provide uniformity of sample gripping and alignment in the instrument. Operator dependence and data variability are greatly reduced.

Conformation Changes, N-terminal Involvement, and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain*

PubMed Central

Wang, Huanchen; Robinson, Howard; Ke, Hengming

2010-01-01

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98–147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes. PMID:20861010

Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Wang; H Robinson; H Ke

2011-12-31

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, whichmore » may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.« less

Influence of clamp-up force on the strength of bolted composite joints

NASA Astrophysics Data System (ADS)

Horn, Walter J.; Schmitt, Ron R.

1994-03-01

Composite materials offer the potential for a reduction in the number of individual parts and joints in a structure because large one-piece components can replace multipart assemblies. Nevertheless, there are many situations where composite parts must be joined and often mechanical fasteners provide the only practical method of joining those parts. The long-term strength of mechanically fastened joints of composite members can be directly affected by the clamp-up force of the fastener and thus perhaps by the relaxation of this force due to the viscoelastic character of the composite materials of the joint. Methods for predicting the effect of bolt clamp-up force relaxation on the strength of mechanically fastened joints of thermoplastic composite materials were investigated during the present study. A test program, using two thermoplastic composite materials, was conducted to determine the influence of clamp-up force on joint strength, to measure the relaxation of the joint clamp-up force with time, and to measure the change of joint strength as a function of time.

33 CFR 183.560 - Hose clamps: Installation.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Hose clamps: Installation. 183.560 Section 183.560 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Manufacturer Requirements § 183.560...

Clamp and Gas Nozzle for TIG Welding

NASA Technical Reports Server (NTRS)

Gue, G. B.; Goller, H. L.

1982-01-01

Tool that combines clamp with gas nozzle is aid to tungsten/inert-gas (TIG) welding in hard-to-reach spots. Tool holds work to be welded while directing a stream of argon gas at weld joint, providing an oxygen-free environment for tungsten-arc welding.

The Anion Paradox in Sodium Taste Reception: Resolution by Voltage-Clamp Studies

NASA Astrophysics Data System (ADS)

Ye, Qing; Heck, Gerard L.; Desimone, John A.

1991-11-01

Sodium salts are potent taste stimuli, but their effectiveness is markedly dependent on the anion, with chloride yielding the greatest response. The cellular mechanisms that mediate this phenomenon are not known. This "anion paradox" has been resolved by considering the field potential that is generated by restricted electrodiffusion of the anion through paracellular shunts between taste-bud cells. Neural responses to sodium chloride, sodium acetate, and sodium gluconate were studied while the field potential was voltage-clamped. Clamping at electronegative values eliminated the anion effect, whereas clamping at electropositive potentials exaggerated it. Thus, field potentials across the lingual epithelium modulate taste reception, indicating that the functional unit of taste reception includes the taste cell and its paracellular microenvironment.

Structure of the Fibrillin-1 N-Terminal Domains Suggests that Heparan Sulfate Regulates the Early Stages of Microfibril Assembly

PubMed Central

Yadin, David A.; Robertson, Ian B.; McNaught-Davis, Joanne; Evans, Paul; Stoddart, David; Handford, Penny A.; Jensen, Sacha A.; Redfield, Christina

2013-01-01

Summary The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly. PMID:24035709

The role of the N-terminal tail for the oligomerization, folding and stability of human frataxin☆

PubMed Central

Faraj, Santiago E.; Venturutti, Leandro; Roman, Ernesto A.; Marino-Buslje, Cristina B.; Mignone, Astor; Tosatto, Silvio C.E.; Delfino, José M.; Santos, Javier

2013-01-01

The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42–80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56–210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81–210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56–81, the moiety putatively responsible for promoting protein–protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56–210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90–210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81–210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function. PMID:23951553

21 CFR 876.5160 - Urological clamp for males.

Code of Federal Regulations, 2010 CFR

2010-04-01

... temporarily. It is an external clamp. (b) Classification. Class I (general controls). Except when intended for internal use or use on females, the device is exempt from the premarket notification procedures in subpart...

Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

PubMed

Singh, Manika Indrajit; Jain, Vikas

2016-01-26

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

Safety evaluation of large external fixation clamps and frames in a magnetic resonance environment.

PubMed

Luechinger, Roger; Boesiger, Peter; Disegi, John A

2007-07-01

Large orthopedic external fixation clamps and related components were evaluated for force, torque, and heating response when subjected to the strong electromagnetic fields of magnetic-resonance (MR) imaging devices. Forces induced by a 3-Tesla (T) MR scanner were compiled for newly designed nonmagnetic clamps and older clamps that contained ferromagnetic components. Heating trials were performed in a 1.5 and in a 3 T MR scanner with two assembled external fixation frames. Forces of the newly designed clamps were more than a factor 2 lower as the gravitational force on the device whereas, magnetic forces on the older devices showed over 10 times the force induced by earth acceleration of gravity. No torque effects could be found for the newly designed clamps. Temperature measurements at the tips of Schanz screws in the 1.5 T MR scanner showed a rise of 0.7 degrees C for a pelvic frame and of 2.1 degrees C for a diamond knee bridge frame when normalized to a specific absorption rate (SAR) of 2 W/kg. The normalized temperature increases in the 3 T MR scanner were 0.9 degrees C for the pelvic frame and 1.1 degrees C for the knee bridge frame. Large external fixation frames assembled with the newly designed clamps (390 Series Clamps), carbon fiber reinforced rods, and implant quality 316L stainless steel Schanz screws met prevailing force and torque limits when tested in a 3-T field, and demonstrated temperature increase that met IEC-60601 guidelines for extremities. The influence of frame-induced eddy currents on the risk of peripheral nerve stimulation was not investigated. Copyright 2006 Wiley Periodicals, Inc.

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, Ursula; Banski, Piotr; Kodiha, Mohamed

2006-08-01

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. Inmore » ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.« less

A peptide N-terminal protection strategy for comprehensive glycoproteome analysis using hydrazide chemistry based method

PubMed Central

Huang, Junfeng; Qin, Hongqiang; Sun, Zhen; Huang, Guang; Mao, Jiawei; Cheng, Kai; Zhang, Zhang; Wan, Hao; Yao, Yating; Dong, Jing; Zhu, Jun; Wang, Fangjun; Ye, Mingliang; Zou, Hanfa

2015-01-01

Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome. PMID:25959593

An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

PubMed

Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

2004-03-01

Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic

Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

PubMed

Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

2013-11-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

PubMed

Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

2016-04-19

Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of sodium chloride on the structure and stability of spider silk’s N-terminal protein domain

PubMed Central

Gronau, Greta; Qin, Zhao; Buehler, Markus J.

2013-01-01

A spider’s ability to store silk protein solutions at high concentration is believed to be related to the protein’s terminal domains. It has been suggested that a shift in salt concentration and pH can have a significant influence on the assembly process. Based on experimental data, a model has been proposed in which the N-terminal domain exists as a monomer during storage and assembles into a homodimer upon spinning. Here we perform a systematic computational study using atomistic, coarse-grained and well-tempered metadynamics simulation to understand how the NaCl concentration in the solution affects the N-terminal domain of the silk protein. Our results show that a high salt concentration, as found during storage, weakens key salt bridges between the monomers, inducing a loss in bond energy by 28.6% in a single salt bridge. As a result dimer formation is less likely as 35.5% less energy is required to unfold the dimer by mechanical force. Conversely, homodimer formation appears to be more likely at low salt concentrations as the salt bridge stays at the lower energy state. The link between salt concentration, structure and stability of the N-terminal domain provides a possible mechanism that prevents premature fiber formation during storage. PMID:23833703

Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation.

PubMed

Shields, Kaitlyn M; Tooley, John G; Petkowski, Janusz J; Wilkey, Daniel W; Garbett, Nichola C; Merchant, Michael L; Cheng, Alan; Schaner Tooley, Christine E

2017-08-01

A subset of B-cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N-terminal trimethylase NRMT1 and the N-terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide-binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N-terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N-terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ-irradiation. © 2017 The Protein Society.

Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

PubMed

Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

2015-01-01

EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

Plasmatic levels of N-terminal pro-atrial natriuretic peptide in preeclamptic patients and healthy normotensive pregnant women.

PubMed

Reyna-Villasmil, Eduardo; Mejia-Montilla, Jorly; Reyna-Villasmil, Nadia; Mayner-Tresol, Gabriel; Herrera-Moya, Pedro; Fernández-Ramírez, Andreina; Rondón-Tapía, Marta

2018-05-11

To compare plasma N-terminal pro-atrial natriuretic peptide concentrations in preeclamptic patients and healthy normotensive pregnant women. A cases-controls study was done with 180 patients at Hospital Central Dr. Urquinaona, Maracaibo, Venezuela, that included 90 preeclamptic patients (group A; cases) and 90 healthy normotensive pregnant women selected with the same age and body mass index similar to group A (group B; controls). Blood samples were collected one hour after admission and prior to administration of any medication in group A to determine plasma N-terminal pro-atrial natriuretic peptide and other laboratory parameters. Plasma N-terminal pro-atrial natriuretic peptide concentrations in group A (mean 1.01 [0.26] pg/mL) showed a significant difference when compared with patients in group B (mean 0.55 [0.07] pg/mL; P<.001]. There was no significant correlation with systolic and diastolic blood pressure values in preeclamptic patients (P=ns). A cut-off value of 0.66ng/mL had an area under the curve of 0.93, sensitivity of 87.8%, specificity of 83.3%, a positive predictive value of 84.0% and a negative predictive value of 87.2%, with a diagnostic accuracy of 85.6%. Preeclamptic patients have significantly higher concentrations of plasma N-terminal pro-atrial natriuretic peptide compared with healthy normotensive pregnant women, with high predictive values for diagnosis. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting.

PubMed

Lin, Pei-Hsuan; Lin, Hsien-Yi; Kuo, Cheng-Chin; Yang, Liang-Tung

2015-06-24

The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on

The optimal protocol to reduce blood loss and blood transfusion after unilateral total knee replacement: Low-dose IA-TXA plus 30-min drain clamping versus drainage clamping for the first 3 h without IA-TXA.

PubMed

Park, Joo Hyun; Choi, Sung Wook; Shin, Eun Ho; Park, Myung Hoon; Kim, Myung Ku

2017-01-01

Although intraarticular tranexamic acid (IA-TXA) administration or drainage clamping are popular methods used to reduce blood loss after total knee replacement (TKR), the protocol remains controversial. We aimed (1) to establish new protocols through investigating whether two methods, that is, low-dose (500 mg) IA-TXA plus 30-min drain clamping and drainage clamping for the first 3 h without IA-TXA, can reduce blood loss and blood transfusion after unilateral TKR and (2) to make recommendations related to clinical application. This study, conducted from September 2014 to June 2016 related to enrolled 95 patients with primary osteoarthritis who were to have a unilateral cemented TKR, was nonrandomized and retrospective. In group A, the drain was released following tourniquet deflation. In group B, 500-mg TXA was injected into the knee joint via a drain tube after fascia closure and the drain was clamped for the first 30 min to prevent leakage. In group C, the drain was clamped for the first 3-h postoperation. Demographic characteristics and clinical data were collected, including the levels of hematocrit (Hct), the total blood loss (TBL), drained blood volume (BV), the amount of blood transfused, and any complications that developed. We found a significantly lower postoperative TBL, drained BV, decreasing Hct level, and less transfused BV in the IA-TXA injection group (group B) and the 3-h drainage clamping group (group C) compared to the conventional negative drainage group (group A; p < 0.001). There was no significant difference between groups B and C ( p = 0.99). The drainage clamping method can be safer than IA-TXA administration in patients with risk factor of venous thromboembolic complication. Furthermore, the IA-TXA administration can be more optimal than drainage clamping in patients with high bleeding tendency or lateral retinacular release during TKR, who would be concerned about postoperative wound complication.

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.

PubMed

Robinson, T; Thomsen, L; Huizinga, J D

2000-05-01

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

CLAMP - a toolkit for efficiently building customized clinical natural language processing pipelines.

PubMed

Soysal, Ergin; Wang, Jingqi; Jiang, Min; Wu, Yonghui; Pakhomov, Serguei; Liu, Hongfang; Xu, Hua

2017-11-24

Existing general clinical natural language processing (NLP) systems such as MetaMap and Clinical Text Analysis and Knowledge Extraction System have been successfully applied to information extraction from clinical text. However, end users often have to customize existing systems for their individual tasks, which can require substantial NLP skills. Here we present CLAMP (Clinical Language Annotation, Modeling, and Processing), a newly developed clinical NLP toolkit that provides not only state-of-the-art NLP components, but also a user-friendly graphic user interface that can help users quickly build customized NLP pipelines for their individual applications. Our evaluation shows that the CLAMP default pipeline achieved good performance on named entity recognition and concept encoding. We also demonstrate the efficiency of the CLAMP graphic user interface in building customized, high-performance NLP pipelines with 2 use cases, extracting smoking status and lab test values. CLAMP is publicly available for research use, and we believe it is a unique asset for the clinical NLP community. © The Author 2017. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

USP48 restrains resection by site-specific cleavage of the BRCA1 ubiquitin mark from H2A.

PubMed

Uckelmann, Michael; Densham, Ruth M; Baas, Roy; Winterwerp, Herrie H K; Fish, Alexander; Sixma, Titia K; Morris, Joanna R

2018-01-15

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.

Effect of complete hilar versus only renal artery clamping on renal histomorphology following ischemia/reperfusion injury in an experimental model.

PubMed

Umul, M; Cal, A C; Turna, B; Oktem, G; Aydın, H H

2016-01-01

To evaluate the effect of temporary complete hilar versus only renal artery clamping with different duration of warm ischemia on renal functions, and possibly identify a "safe" clamping type and duration of renal ischemia. Fifty male rabbits have been incorporated to study. Rabbits were subjected to ischemia/reperfusion injury by temporary vascular clamping. Reagents were randomized to 3 experimental groups (only renal artery clamping, complete hilar clamping, sham surgery) and sub-groups were determined according to different clamping times (30 and 60 minutes). Median laparotomy and left renal hilus dissection were performed to sham group. Only artery or complete hilar clamping was performed for 30 or 60 minutes by microvascular bulldog clamps to other reagents. Rabbits were sacrificed 10 days after primary surgery and left nephrectomy performed. Nephrectomy materials were evaluated for the level of nitric-oxide synthase (NOS) immunoreactivity, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity and an electron microscopic examination was performed. NOS immunoreactivity was correlated with the temporary clamping time. We also observed that complete hilar vascular clamping entails an increase on NOS immunoreactivity. MDA levels were similar for all experimental surgery groups (p = 0.42). The SOD activity was decreased among all subgroups compared with sham surgery. But the significant decrease occurred in 30 minutes only artery and 30 minutes complete hilar clamping groups in proportion to sham surgery (p = 0.026 and p = 0.019, respectively). This current study suggested that only renal artery clamping under 30 minutes is more appropriate during renal surgical procedures requiring temporary vascular clamping.

Resonant-type Smooth Impact Drive Mechanism (SIDM) actuator using a bolt-clamped Langevin transducer.

PubMed

Nishimura, Takuma; Hosaka, Hiroshi; Morita, Takeshi

2012-01-01

The Smooth Impact Drive Mechanism (SIDM) is a linear piezoelectric actuator that has seen practically applied to camera lens modules. Although previous SIDM actuators are easily miniaturized and enable accurate positioning, these actuators cannot actuate at high speed and cannot provide powerful driving because they are driven at an off-resonant frequency using a soft-type PZT. In the present study, we propose a resonant-type SIDM using a bolt-clamped Langevin transducer (BLT) with a hard-type PZT. The resonant-type SIDM overcomes the above-mentioned problems and high-power operation becomes possible with a very simple structure. As a result, we confirmed the operation of resonant-type SIDM by designing a bolt-clamped Langevin transducer. The properties of no-load maximum speed was 0.28m/s at driving voltages of 80V(p-p) for 44.9kHz and 48V(p-p) for 22.45kHz with a pre-load of 3.1N. Copyright © 2011 Elsevier B.V. All rights reserved.

Nitrogen termination of single crystal (100) diamond surface by radio frequency N{sub 2} plasma process: An in-situ x-ray photoemission spectroscopy and secondary electron emission studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandran, Maneesh, E-mail: maneesh@tx.technion.ac.il, E-mail: choffman@tx.technion.ac.il; Shasha, Michal; Michaelson, Shaul

2015-09-14

In this letter, we report the electronic and chemical properties of nitrogen terminated (N-terminated) single crystal (100) diamond surface, which is a promising candidate for shallow NV{sup −} centers. N-termination is realized by an indirect RF nitrogen plasma process without inducing a large density of surface defects. Thermal stability and electronic property of N-terminated diamond surface are systematically investigated under well-controlled conditions by in-situ x-ray photoelectron spectroscopy and secondary electron emission. An increase in the low energy cut-off of the secondary electron energy distribution curve (EDC), with respect to a bare diamond surface, indicates a positive electron affinity of themore » N-terminated diamond. Exposure to atomic hydrogen results in reorganization of N-terminated diamond to H-terminated diamond, which exhibited a negative electron affinity surface. The change in intensity and spectral features of the secondary electron EDC of the N-terminated diamond is discussed.« less

Synthetic polymers as substrates for a DNA-sliding clamp protein.

PubMed

van Dongen, S F M; Clerx, J; van den Boomen, O I; Pervaiz, M; Trakselis, M A; Ritschel, T; Schoonen, L; Schoenmakers, D C; Nolte, R J M

2018-04-26

The clamp protein (gp45) of the DNA polymerase III of the bacteriophage T4 is known to bind to DNA and stay attached to it in order to facilitate the process of DNA copying by the polymerase. As part of a project aimed at developing new biomimetic data-encoding systems we have investigated the binding of gp45 to synthetic polymers, that is, rigid, helical polyisocyanopeptides. Molecular modelling studies suggest that the clamp protein may interact with the latter polymers. Experiments aimed at verifying these interactions are presented and discussed. © 2018 The Authors Biopolymers Published by Wiley Periodicals, Inc.

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

PubMed Central

Lelj-Garolla, Barbara; Mauk, A Grant

2012-01-01

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1–14 and Δ1–24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association. PMID:22057845

An implementation of the maximum-caliber principle by replica-averaged time-resolved restrained simulations

NASA Astrophysics Data System (ADS)

Capelli, Riccardo; Tiana, Guido; Camilloni, Carlo

2018-05-01

Inferential methods can be used to integrate experimental informations and molecular simulations. The maximum entropy principle provides a framework for using equilibrium experimental data, and it has been shown that replica-averaged simulations, restrained using a static potential, are a practical and powerful implementation of such a principle. Here we show that replica-averaged simulations restrained using a time-dependent potential are equivalent to the principle of maximum caliber, the dynamic version of the principle of maximum entropy, and thus may allow us to integrate time-resolved data in molecular dynamics simulations. We provide an analytical proof of the equivalence as well as a computational validation making use of simple models and synthetic data. Some limitations and possible solutions are also discussed.

An implementation of the maximum-caliber principle by replica-averaged time-resolved restrained simulations.

PubMed

Capelli, Riccardo; Tiana, Guido; Camilloni, Carlo

2018-05-14

Inferential methods can be used to integrate experimental informations and molecular simulations. The maximum entropy principle provides a framework for using equilibrium experimental data, and it has been shown that replica-averaged simulations, restrained using a static potential, are a practical and powerful implementation of such a principle. Here we show that replica-averaged simulations restrained using a time-dependent potential are equivalent to the principle of maximum caliber, the dynamic version of the principle of maximum entropy, and thus may allow us to integrate time-resolved data in molecular dynamics simulations. We provide an analytical proof of the equivalence as well as a computational validation making use of simple models and synthetic data. Some limitations and possible solutions are also discussed.

Investigation on frictional characteristics and drawbead restraining force of steel with/without coating

NASA Astrophysics Data System (ADS)

Chen, Lianfeng; Zheng, Tianran; Chen, Qing; Zhang, Jun

2013-12-01

Advanced high strength steels (AHSS) are used more and more in automotive industry for increasing crashworthiness and weight reduction. Improving metal flow and reduce friction are important to forming the part and decrease part reject rates of AHSS. The present study focused on friction characteristics and drawbead restraining force of Dual Phase (DP) steels with or without coating, such as DP980, DP780, DP590, DP780+Z, DP780+ZF, DP590+Z, using experimental approach. The effect of material properties, temperature, sliding velocity, surface roughness, dry and lubricant on friction behavior of DP steels is investigated. The contrast of DP steels with mild IF steel is carried out. The restraining force draw through different radius of drawbead is evaluated. This study is benefit to the set up of technique parameters during sheet metal forming simulation.

A novel way to go whole-cell in patch-clamp experiments.

PubMed

Inayat, Samsoon; Zhao, Yan; Cantrell, Donal R; Dikin, Dmitryi; Pinto, Lawrence H; Troy, John B

2010-11-01

With a conventional patch-clamp electrode, an Ag/AgCl wire sits stationary inside the pipette. To move from the gigaseal cell-attached configuration to whole-cell recording, suction is applied inside the pipette. We have designed and developed a novel Pushpen patch-clamp electrode, in which a W wire insulated and wound with Ag/AgCl wire can move linearly inside the pipette. The W wire has a conical tip, which can protrude from the pipette tip like a push pen, a procedure we call the Pushpen Operation. We use the Pushpen operation to impale the cell membrane in cell-attached configuration to go whole-cell without disruption of the gigaseal. We successfully recorded whole-cell currents from chinese hamster ovarian cells expressing influenza A virus protein A/M2, after obtaining whole-cell configuration with the Pushpen operation. This novel method of achieving whole-cell configuration may have a higher success rate than is the case with the conventional patch clamp technique.

Randomized clinical trial of stapler versus clamp-crushing transection in elective liver resection.

PubMed

Rahbari, N N; Elbers, H; Koch, M; Vogler, P; Striebel, F; Bruckner, T; Mehrabi, A; Schemmer, P; Büchler, M W; Weitz, J

2014-02-01

Various devices have been developed to facilitate liver transection and reduce blood loss in liver resections. None of these has proven superiority compared with the classical clamp-crushing technique. This randomized clinical trial compared the effectiveness and safety of stapler transection with that of clamp-crushing during open liver resection. Patients admitted for elective open liver resection between January 2010 and October 2011 were assigned randomly to stapler transection or the clamp-crushing technique. The primary endpoint was the total amount of intraoperative blood loss. Secondary endpoints included transection time, duration of operation, complication rates and resection margins. A total of 130 patients were enrolled, 65 to clamp-crushing and 65 to stapler transection. There was no difference between groups in total intraoperative blood loss: median (i.q.r.) 1050 (525-1650) versus 925 (450-1425) ml respectively (P = 0·279). The difference in total intraoperative blood loss normalized to the transection surface area was not statistically significant (P = 0·092). Blood loss during parenchymal transection was significantly lower in the stapler transection group (P = 0·002), as were the parenchymal transection time (mean(s.d.) 30(21) versus 9(7) min for clamp-crushing and stapler transection groups respectively; P < 0·001) and total duration of operation (mean(s.d.) 221(86) versus 190(85) min; P = 0·047). There were no significant differences in postoperative morbidity (P = 0·863) or mortality (P = 0·684) between groups. Stapler transection is a safe technique but does not reduce intraoperative blood loss in elective liver resection compared with the clamp-crushing technique. NCT01049607 (http://www.clinicaltrials.gov). © 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.

Substrate clamping effects on irreversible domain wall dynamics in lead zirconate titanate thin films.

PubMed

Griggio, F; Jesse, S; Kumar, A; Ovchinnikov, O; Kim, H; Jackson, T N; Damjanovic, D; Kalinin, S V; Trolier-McKinstry, S

2012-04-13

The role of long-range strain interactions on domain wall dynamics is explored through macroscopic and local measurements of nonlinear behavior in mechanically clamped and released polycrystalline lead zirconate-titanate (PZT) films. Released films show a dramatic change in the global dielectric nonlinearity and its frequency dependence as a function of mechanical clamping. Furthermore, we observe a transition from strong clustering of the nonlinear response for the clamped case to almost uniform nonlinearity for the released film. This behavior is ascribed to increased mobility of domain walls. These results suggest the dominant role of collective strain interactions mediated by the local and global mechanical boundary conditions on the domain wall dynamics. The work presented in this Letter demonstrates that measurements on clamped films may considerably underestimate the piezoelectric coefficients and coupling constants of released structures used in microelectromechanical systems, energy harvesting systems, and microrobots.

Constant-frequency, clamped-mode resonant converters

NASA Technical Reports Server (NTRS)

Tsai, Fu-Sheng; Materu, Peter; Lee, Fred C.

1987-01-01

Two novel clamped-mode resonant converters are proposed which operate at a constant frequency while retaining many desired features of conventional series- and parallel-resonant converters. State-plane analysis techniques are used to identify all possible operating modes and define their mode boundaries. Control-to-output characteristics are derived that specify the regions for natural and forced commutation. The predicted operating modes are verified using a prototype circuit.

The structure of S . lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain

DOE PAGES

Mitchell, Carter A.; Tucker, Alex C.; Escalante-Semerena, Jorge C.; ...

2014-12-09

The adenosine monoposphate-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ~110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. In this paper, the structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to othermore » members of this family. Finally, whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain.« less

CO adsorption on small Au{sub n} (n = 1–4) structures supported on hematite. II. Adsorption on the O-rich termination of α-Fe{sub 2}O{sub 3}(0001) surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pabisiak, Tomasz; Kiejna, Adam, E-mail: kiejna@ifd.uni.wroc.pl; Winiarski, Maciej J.

2016-01-28

The adsorption of small Au{sub n} (n = 1–4) nanostructures on oxygen terminated α-Fe{sub 2}O{sub 3}(0001) surface was investigated using density functional theory in the generalized gradient approximation of Perdew-Burke-Ernzerhof (PBE) form with Hubbard correction U, accounting for strong electron correlations (PBE+U). The structural, energetic, and electronic properties were examined for two classes of the adsorbed Au{sub n} nanostructures with vertical and flattened configurations. Similarly to the Fe-terminated α-Fe{sub 2}O{sub 3}(0001) surface considered in Part I, the flattened configurations were found energetically more favored than vertical ones. The binding of Au{sub n} to the O-terminated surface is much stronger thanmore » to the Fe-termination. The adsorption bonding energy of Au{sub n} and the work function of the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) systems decrease with the increased number of Au atoms in a structure. All of the adsorbed Au{sub n} structures are positively charged. The bonding of CO molecules to the Au{sub n} structures is distinctly stronger than on the Fe-terminated surface; however, it is weaker than the binding to the bare O-terminated surface. The CO molecule binds to the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) system through a peripheral Au atom partly detached from the Au{sub n} structure. The results of this work indicate that the most energetically favored sites for adsorption of a CO molecule on the Au{sub n}/α-Fe{sub 2}O{sub 3}(0001) systems are atoms in the Au{sup 0.5+} oxidation state.« less

Thermal Behavior of Cylindrical Buckling Restrained Braces at Elevated Temperatures

PubMed Central

Talebi, Elnaz; Tahir, Mahmood Md.; Yasreen, Airil

2014-01-01

The primary focus of this investigation was to analyze sequentially coupled nonlinear thermal stress, using a three-dimensional model. It was meant to shed light on the behavior of Buckling Restraint Brace (BRB) elements with circular cross section, at elevated temperature. Such bracing systems were comprised of a cylindrical steel core encased in a strong concrete-filled steel hollow casing. A debonding agent was rubbed on the core's surface to avoid shear stress transition to the restraining system. The numerical model was verified by the analytical solutions developed by the other researchers. Performance of BRB system under seismic loading at ambient temperature has been well documented. However, its performance in case of fire has yet to be explored. This study showed that the failure of brace may be attributed to material strength reduction and high compressive forces, both due to temperature rise. Furthermore, limiting temperatures in the linear behavior of steel casing and concrete in BRB element for both numerical and analytical simulations were about 196°C and 225°C, respectively. Finally it is concluded that the performance of BRB at elevated temperatures was the same as that seen at room temperature; that is, the steel core yields prior to the restraining system. PMID:24526915

Thermal behavior of cylindrical buckling restrained braces at elevated temperatures.

PubMed

Talebi, Elnaz; Tahir, Mahmood Md; Zahmatkesh, Farshad; Yasreen, Airil; Mirza, Jahangir

2014-01-01

The primary focus of this investigation was to analyze sequentially coupled nonlinear thermal stress, using a three-dimensional model. It was meant to shed light on the behavior of Buckling Restraint Brace (BRB) elements with circular cross section, at elevated temperature. Such bracing systems were comprised of a cylindrical steel core encased in a strong concrete-filled steel hollow casing. A debonding agent was rubbed on the core's surface to avoid shear stress transition to the restraining system. The numerical model was verified by the analytical solutions developed by the other researchers. Performance of BRB system under seismic loading at ambient temperature has been well documented. However, its performance in case of fire has yet to be explored. This study showed that the failure of brace may be attributed to material strength reduction and high compressive forces, both due to temperature rise. Furthermore, limiting temperatures in the linear behavior of steel casing and concrete in BRB element for both numerical and analytical simulations were about 196°C and 225°C, respectively. Finally it is concluded that the performance of BRB at elevated temperatures was the same as that seen at room temperature; that is, the steel core yields prior to the restraining system.

Associations between Restrained Eating and the Size and Frequency of Overall Intake, Meal, Snack and Drink Occasions in the UK Adult National Diet and Nutrition Survey

PubMed Central

Olea López, Ana Lorena; Johnson, Laura

2016-01-01

Obesity is a global public health priority. Restrained eating is related to obesity and total energy intake but associations with the eating patterns are unclear. We examined the associations of restrained eating with the size and frequency of intake occasions among 1213 British adult (19–64 y) participants in a cross-sectional analysis of the UK National Diet and Nutrition Survey 2000. The Dutch Eating Behaviour Questionnaire assessed restrained eating. Overall intake occasions were all energy consumed in a 60 min period. A food-based classification separated intake occasions into meals, snacks, or drinks from seven-day weighed food diaries. Average daily frequency and size (kcal) of overall intake, meal, snack and drink occasions were calculated and associations with restrained eating were modelled using multiple linear regression including under-reporting of energy intake, age, gender, BMI, emotional eating, external eating and physical activity as covariates. Restrained eating was very weakly positively correlated with overall intake (r = 0.08, p<0.05) and meal frequency (r = 0.10, p<0.05) but not snack or drink frequency (r = 0.02 and -0.02 respectively). Adjusted regressions showed a one-point change in restrained eating was associated with 0.07 (95% CI 0.03, 0.11) more meal occasions/day and 0.13 (95% CI 0.01, 0.25) extra overall intake occasions/day. Overall intake occasion size was weakly negatively correlated with restrained eating regardless of type (r = -0.16 to -0.20, all p<0.0001). Adjusted regressions showed each one-point increase in restrained eating was associated with lower-energy meals (-15 kcal 95% CI -5.9, -24.2) and drinks (-4 kcal 95%CI -0.1, -8), but not snacks or overall intake occasions. Among a national sample of UK adults, greater restrained eating was associated with smaller and slightly more frequent eating, suggesting that restrained eaters restrict their energy intake by reducing meal/drink size rather than skipping snacks. PMID

Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

PubMed Central

Folio, Christelle; Sierra, Natalia; Dujardin, Marie; Alvarez, Guzman

2017-01-01

Feline immunodeficiency virus (FIV) is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS) in cats and wild felines. Its capsid protein (CA) drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA. PMID:29120364

Artery cross-clamping during laparoscopic vascular surgeries; a computational tactile sensing approach.

PubMed

Pahlavan, Pedram; Najarian, Siamak; Afshari, Elnaz; Moini, Majid

2013-01-01

Artificial palpation is one of the most valuable achievements of artificial tactile sensing approach that can be used in various fields of medicine and more specifically in surgery. These techniques cause different surgical maneuvers to be done more precisely and noninvasively. In this study, considering the present problems and limitations of cross-clamping an artery during laparoscopic vascular surgeries, a new tactile sensory system will be introduced.Having imitated surgeon's palpation during open vascular surgeries and modeled it conceptually, the optimal amount of the total angular displacement of each robot joint in order to cross-clamping an artery without damaging to the artery surrounding tissues will be calculated. The elastic governing equation of contact occurred between the tactile sensor placed on the first link of the robot and the surrounding tissues around the artery were developed. A finite element model is coupled with genetic algorithm optimization method so that the normal stress and displacements in contact surface of the robot and artery's surrounding tissues would be minimized. Thus, reliability and accuracy of artificial tactile sensing method in artery cross-clamping will be demonstrated. Finally, the functional principles of the new tactile system capable of cross-clamping an artery during laparoscopic surgeries will be presented.

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas

Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 thatmore » forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.« less

Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

PubMed Central

Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

2013-01-01

In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

Efficacy of tranexamic acid plus drain-clamping to reduce blood loss in total knee arthroplasty: A meta-analysis.

PubMed

Zhang, Yan; Zhang, Jun-Wei; Wang, Bao-Hua

2017-06-01

Perioperative blood loss is still an unsolved problem in total knee arthroplasty (TKA). The efficacy of the preoperative use of tranexamic acid (TXA) plus drain-clamping to reduce blood loss in TKA has been debated. This meta-analysis aimed to illustrate the efficacy of TXA plus drain-clamping to reduce blood loss in patients who underwent a TKA. In February 2017, a systematic computer-based search was conducted in PubMed, EMBASE, Web of Science, the Cochrane Database of Systematic Reviews, and Google Scholar. Data from patients prepared for TKA in studies that compared TXA plus drain-clamping versus TXA alone, drain-clamping alone, or controls were retrieved. The primary endpoint was the need for transfusion. The secondary outcomes were total blood loss, blood loss in drainage, the decrease in hemoglobin, and the occurrence of deep venous thrombosis. After testing for publication bias and heterogeneity between studies, data were aggregated for random-effects models when necessary. Ultimately, 5 clinical studies with 618 patients (TXA plus drain-clamping group = 249, control group = 130, TXA-alone group = 60, and drain-clamping group = 179) were included. TXA plus drain-clamping could decrease the need for transfusion, total blood loss, blood loss in drainage, and the decrease in hemoglobin than could the control group, the TXA-alone group, and the drain-clamping group (P < .05). There was no significant difference between the occurrence of deep venous thrombosis between the included groups (P > .05). TXA plus drain-clamping can achieve the maximum effects of hemostasis in patients prepared for primary TKA. Because the number and the quality of the included studies were limited, more high-quality randomized controlled trials are needed to identify the optimal dose of TXA and the clamping hours in patients prepared for TKA.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

PubMed

Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

N-TERMINALLY ELONGATED SpliInx2 AND SpliInx3 REDUCE BACULOVIRUS-TRIGGERED APOPTOSIS VIA HEMICHANNEL CLOSURE.

PubMed

Chen, Ya-Bin; Xiao, Wei; Li, Ming; Zhang, Yan; Yang, Yang; Hu, Jian-Sheng; Luo, Kai-Jun

2016-05-01

The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate. An insect Bac-to-Bac expression system, pFastBac(TM) HT A, allows us to construct an N-terminally elongated SpliInx2 (Nte-Inx2) and SpliInx3 (Nte-Inx3). Here, we demonstrated that recombinant baculovirus Bac-Nte-Inx2 (reBac-Net-Inx2) and Bac-Nte-Inx3 (reBac-Nte-Inx3) closed the endogenous hemichannel on the Sf9 cell surface. Importantly, primary baculovirus infections significantly caused early apoptosis, and this apoptosis was reduced by hemichannel-closed Sf9 cells at 24-h post-infection (PI). Although N-terminal-elongated residue led to the increase in the phosphorylated sites in both Nte-Inx2 and Nte-Inx3 and an additional transmembrane domain in Nte-Inx3, both the proteins localized on the cell surface, suggesting Nte-Inxs proteins could mediate hemichannel closure. Further supporting evidence showed that hemichannel closure was dependent on N-Inxs expressed by baculovirus polyhedrin promoter, which began to express at 18-24 h PI. These results identify an unconventional function of N-terminal-elongated innexins that could act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from intracellular to extracellular compartment. © 2016 Wiley Periodicals, Inc.

Relationship of Dieting and Restrained Eating to Self-Reported Caloric Intake in Female College Freshmen

PubMed Central

Goldstein, Stephanie P.; Katterman, Shawn N.; Lowe, Michael R.

2012-01-01

Evidence indicates that restrained eaters do not eat less than unrestrained eaters in the natural environment. However, no study has examined caloric intake in those who are currently dieting to lose, or avoid gaining, weight. The current study examined caloric intake using 24-hour food recalls among individuals dieting to lose weight, dieting to avoid weight gain, restrained nondieters, and unrestrained nondieters. Participants were 246 female college students participating in a weight gain prevention trial. The predicted significant difference in caloric intake across the four groups was found for beverage but not for food intake. Results reinforce past literature indicating that dieting/restraint status does not reflect hypo-caloric intake in naturalistic settings. PMID:23557829

Expression, crystallization and preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins from Thermoplasma acidophilum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Sang Hee; Ha, Jun Yong; Kim, Kyoung Hoon

2006-11-01

An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively. N-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring in approximately 80–90% of cytosolic mammalian proteins and about 50% of yeast proteins. ARD1 (arrest-defective protein 1), together with NAT1 (N-acetyltransferase protein 1) and possibly NAT5, is responsible for the NatA activity in Saccharomyces cerevisiae. In mammals, ARD1 is involved in cell proliferation, neuronal development and cancer. Interestingly, it has been reported that mouse ARD1 (mARD1{sup 225}) mediatesmore » ∊-acetylation of hypoxia-inducible factor 1α (HIF-1α) and thereby enhances HIF-1α ubiquitination and degradation. Here, the preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins encoded by the Ta0058 and Ta1140 genes of Thermoplasma acidophilum are reported. The Ta0058 protein is related to an N-terminal acetyltransferase complex ARD1 subunit, while Ta1140 is a putative N-terminal acetyltransferase-related protein. Ta0058 shows 26% amino-acid sequence identity to both mARD1{sup 225} and human ARD1{sup 235}.The sequence identity between Ta0058 and Ta1140 is 28%. Ta0058 and Ta1140 were overexpressed in Escherichia coli fused with an N-terminal purification tag. Ta0058 was crystallized at 297 K using a reservoir solution consisting of 0.1 M sodium acetate pH 4.6, 8%(w/v) polyethylene glycol 4000 and 35%(v/v) glycerol. X-ray diffraction data were collected to 2.17 Å. The Ta0058 crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 49.334, c = 70.384 Å, α = β = γ = 90°. The asymmetric unit contains a monomer, giving a calculated crystal volume per protein weight (V{sub M}) of 2.13 Å{sup 3} Da{sup −1} and a solvent

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.

PubMed

Sakuma, Chisato; Sato, Mitsuru; Takenouchi, Takato; Kitani, Hiroshi

2015-02-01

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

U.S. Transit Track Restraining Rail. Volume I : Study of Requirements and Practices

DOT National Transportation Integrated Search

1981-12-01

This report covers a study of restraining rails in transit track, which is part of the current research program of UMTA and was initiated: (1) to assist in the analysis, design, and maintenance and operation of transit track; (2) to compile guideline...

Measurement and modeling of intrinsic transcription terminators

PubMed Central

Cambray, Guillaume; Guimaraes, Joao C.; Mutalik, Vivek K.; Lam, Colin; Mai, Quynh-Anh; Thimmaiah, Tim; Carothers, James M.; Arkin, Adam P.; Endy, Drew

2013-01-01

The reliable forward engineering of genetic systems remains limited by the ad hoc reuse of many types of basic genetic elements. Although a few intrinsic prokaryotic transcription terminators are used routinely, termination efficiencies have not been studied systematically. Here, we developed and validated a genetic architecture that enables reliable measurement of termination efficiencies. We then assembled a collection of 61 natural and synthetic terminators that collectively encode termination efficiencies across an ∼800-fold dynamic range within Escherichia coli. We simulated co-transcriptional RNA folding dynamics to identify competing secondary structures that might interfere with terminator folding kinetics or impact termination activity. We found that structures extending beyond the core terminator stem are likely to increase terminator activity. By excluding terminators encoding such context-confounding elements, we were able to develop a linear sequence-function model that can be used to estimate termination efficiencies (r = 0.9, n = 31) better than models trained on all terminators (r = 0.67, n = 54). The resulting systematically measured collection of terminators should improve the engineering of synthetic genetic systems and also advance quantitative modeling of transcription termination. PMID:23511967

Improving cell penetration of helical peptides stabilized by N-terminal crosslinked aspartic acids.

PubMed

Zhao, Hui; Jiang, Yanhong; Tian, Yuan; Yang, Dan; Qin, Xuan; Li, Zigang

2017-01-04

Cell penetration and nucleus translocation efficiency are important for the cellular activities of peptide therapeutics. For helical peptides stabilized by N-terminal crosslinked aspartic acid, correlations between their penetration efficiency/nucleus translocation and physicochemical properties were studied. An increase in hydrophobicity and isoelectric point will promote cellular uptake and nucleus translocation of stabilized helices.

Long-Time Choledochal Clamping in Wistar Rats Causes Biliary Obstruction Progressing to Hepatic Fibrosis.

PubMed

Jorge, G D L; Tártaro, R R; Escanhoela, C A F; Boin, I D F S F

2016-09-01

Biliary complications are important causes of morbidity and mortality in patients undergoing hepatic surgery. The aim of the study was to evaluate late liver alterations after a long period of choledochal clamping in Wistar rats. Ten male Wistar rats, weighing 304 grams, anesthetized with sodium thiopental (25 mg/kg) and xylazine (10 mg/kg) intravenously, were distributed into 2 groups: the choledochal clamping group (CCG) and the operation sham group (OSG), with 5 animals each submitted to an abdominal incision. In the CCG, the choledochal was isolated, dissected, and clamped with a microvascular clamp for 40 minutes. After this occlusion time, the clamp was removed and the incision was closed. In the OSG the animals, under normal conditions, were submitted only to anesthesia and laparotomy for choledochal manipulation. In all animals, after the 31st day, a hepatic biopsy was carried out for histology and blood biochemical tests: total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase. The animals were euthanized under anesthesia. This research was approved by the Ethics Committee on Animal Use (CEUA, Unicamp, No. 2511-1). In the CCG, 100% of the animals showed bile duct dilatation, ductular proliferation, and portal inflammatory infiltrate; 60% showed regenerative nodule formation; and 80% had porta-porta septa and foci of necrosis, all of which were not found in the OSG. All CCG group biochemical tests had significant increases (P < .05) compared with OSG. Long-time choledochal clamping in Wistar rats caused hepatic dysfunction and biochemical and histological injuries with degrees of distortion to the hepatic architecture. Copyright © 2016 Elsevier Inc. All rights reserved.

Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes.

PubMed

Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P; Veldkamp, Christopher T

2016-01-01

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis. © 2016 Elsevier Inc. All rights reserved.

Preparation and analysis of N-terminal chemokine receptor sulfopeptides using tyrosylprotein sulfotransferase enzymes

PubMed Central

Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P.; Veldkamp, Christopher T.

2016-01-01

In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by post-translational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8 and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the liability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods from sulfopeptide analysis. PMID:26921955

Collision-avoidance behaviors of minimally restrained flying locusts to looming stimuli

PubMed Central

Chan, R. WM.; Gabbiani, F.

2013-01-01

SUMMARY Visually guided collision avoidance is of paramount importance in flight, for instance to allow escape from potential predators. Yet, little is known about the types of collision-avoidance behaviors that may be generated by flying animals in response to an impending visual threat. We studied the behavior of minimally restrained locusts flying in a wind tunnel as they were subjected to looming stimuli presented to the side of the animal, simulating the approach of an object on a collision course. Using high-speed movie recordings, we observed a wide variety of collision-avoidance behaviors including climbs and dives away from – but also towards – the stimulus. In a more restrained setting, we were able to relate kinematic parameters of the flapping wings with yaw changes in the trajectory of the animal. Asymmetric wing flapping was most strongly correlated with changes in yaw, but we also observed a substantial effect of wing deformations. Additionally, the effect of wing deformations on yaw was relatively independent of that of wing asymmetries. Thus, flying locusts exhibit a rich range of collision-avoidance behaviors that depend on several distinct aerodynamic characteristics of wing flapping flight. PMID:23364572

N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisone, G.; Berthold, M.; Bedecs, K.

1989-12-01

The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis.more » Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.« less

N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803

DOE PAGES

Juneau, Andrea D.; Frankel, Laurie K.; Bricker, Terry M.; ...

2016-09-22

Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 tomore » eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less

N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juneau, Andrea D.; Frankel, Laurie K.; Bricker, Terry M.

Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 tomore » eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PubMed

Ramkumar, Srinivasagan; Fujii, Noriko; Fujii, Norihiko; Thankappan, Bency; Sakaue, Hiroaki; Ingu, Kim; Natarajaseenivasan, Kalimuthusamy; Anbarasu, Kumarasamy

2014-01-01

To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed β-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

SCBUCKLE user's manual: Buckling analysis program for simple supported and clamped panels

NASA Technical Reports Server (NTRS)

Cruz, Juan R.

1993-01-01

The program SCBUCKLE calculates the buckling loads and mode shapes of cylindrically curved, rectangular panels. The panel is assumed to have no imperfections. SCBUCKLE is capable of analyzing specially orthotropic symmetric panels (i.e., A(sub 16) = A(sub 26) = 0.0, D(sub 16) = D(sub 26) = 0.0, B(sub ij) = 0.0). The analysis includes first-order transverse shear theory and is capable of modeling sandwich panels. The analysis supports two types of boundary conditions: either simply supported or clamped on all four edges. The panel can be subjected to linearly varying normal loads N(sub x) and N(sub y) in addition to a constant shear load N(sub xy). The applied loads can be divided into two parts: a preload component; and a variable (eigenvalue-dependent) component. The analysis is based on the modified Donnell's equations for shallow shells. The governing equations are solved by Galerkin's method.

30 CFR 18.40 - Cable clamps and grips.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Cable clamps and grips. 18.40 Section 18.40 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR TESTING, EVALUATION, AND APPROVAL OF MINING PRODUCTS ELECTRIC MOTOR-DRIVEN MINE EQUIPMENT AND ACCESSORIES Construction and Design...

Deglacial hydrography and IRD inputs: A comparison of Terminations I and II in the N.E. Atlantic

NASA Astrophysics Data System (ADS)

Hibbert, Fiona; Chapman, Mark; Austin, William; Rohling, Eelco

2015-04-01

We present a high resolution marine record (MD04-2822) from the N.E. Atlantic. This record captures the demise of the penultimate glaciation (Termination II) in high resolution. The record of co-registered proxies offers the opportunity to investigate the evolution of the last two deglacial events in the North Atlantic. The deglacial evolution of Termination II is much less well documented than the last deglaciation (Termination I). A striking feature of Termination II in the MD04-2822 record, are several large (~1 ‰) oscillations in benthic δ18O, reflecting oscillations in sea level (e.g. Grant et al., 2012, Thomas et al., 2009) and/or deep sea temperatures (cf. Skinner and Shackleton, 2006). Also notable is the markedly different pattern of surface and deep water evolution for the two deglaciations. Termination I is characterised by a short offset between benthic δ18O decrease and δ13C increase (and northwards migration of the polar front) whereas during Termination II, benthic δ13C 'improvement' (and inferred resumption in overturning) occurs only during the Marine Isotope Stage (MIS) 5e plateau, giving the marine record it's characteristic 'drawn-out' appearance. The most conspicuous feature of the penultimate deglacial in most marine cores is Heinrich event 11 (H11), an extensive episode of ice rafted debris (IRD) discharge that spread across the North Atlantic to the margin of what is now the subtropical gyre (Chapman et al., 2000). H11 generally manifests in marine records as one large and long (~ 2.5 ka) event throughout the Termination. In MD04-2822 however, there are multiple IRD events within the Termination. The continued influence of the disintegrating N. hemisphere ice sheets is also evident within the benthic δ13C and surface conditions (the polar front migrates north of the core site early within MIS 5e following a brief SST reversal).

33 CFR 183.532 - Clips, straps, and hose clamps.

Code of Federal Regulations, 2010 CFR

2010-07-01

... (CONTINUED) BOATING SAFETY BOATS AND ASSOCIATED EQUIPMENT Fuel Systems Equipment Standards § 183.532 Clips... under § 183.590, a hose clamp installed on a fuel line system requiring metallic fuel lines or “USCG...

Functional hierarchy of the N-terminal tyrosines of SLP-76.

PubMed

Jordan, Martha S; Sadler, Jeffrey; Austin, Jessica E; Finkelstein, Lisa D; Singer, Andrew L; Schwartzberg, Pamela L; Koretzky, Gary A

2006-02-15

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a central role in T cell activation and T cell development. SLP-76 has three functional modules: an acidic domain with three key tyrosines, a central proline-rich domain, and a C-terminal Src homology 2 domain. Of these, mutation of the three N-terminal tyrosines (Y112, Y128, and Y145) results in the most profound effects on T cell development and function. Y112 and Y128 associate with Vav and Nck, two proteins shown to be important for TCR-induced phosphorylation of proximal signaling substrates, Ca(2+) flux, and actin reorganization. Y145 has been shown to be important for optimal association of SLP-76 with inducible tyrosine kinase, a key regulator of T cell function. To investigate further the role of the phosphorylatable tyrosines of SLP-76 in TCR signaling, cell lines and primary T cells expressing SLP-76 with mutations in individual or paired tyrosine residues were analyzed. These studies show that Tyr(145) of SLP-76 is the most critical tyrosine for both T cell function in vitro and T cell development in vivo.

Role of the α clamp in the protein translocation mechanism of anthrax toxin

PubMed Central

Brown, Michael J.; Thoren, Katie L.; Krantz, Bryan A.

2015-01-01

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins—protective antigen (PA), lethal factor (LF), and edema factor (EF). Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds LF and EF and translocates them into the host cytosol. Translocation is driven by the proton motive force, comprised of the chemical potential, the proton-gradient (ΔpH), and the membrane potential (ΔΨ). A crystal structure of the lethal toxin core complex revealed an “α clamp” structure that binds to substrate helices nonspecifically. Here we test the hypothesis that through the recognition of unfolding helical structure the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation. PMID:26344833

Thermodynamic basis for engineering high-affinity, high-specificity binding-induced DNA clamp nanoswitches.

PubMed

Idili, Andrea; Plaxco, Kevin W; Vallée-Bélisle, Alexis; Ricci, Francesco

2013-12-23

Naturally occurring chemoreceptors almost invariably employ structure-switching mechanisms, an observation that has inspired the use of biomolecular switches in a wide range of artificial technologies in the areas of diagnostics, imaging, and synthetic biology. In one mechanism for generating such behavior, clamp-based switching, binding occurs via the clamplike embrace of two recognition elements onto a single target molecule. In addition to coupling recognition with a large conformational change, this mechanism offers a second advantage: it improves both affinity and specificity simultaneously. To explore the physics of such switches we have dissected here the thermodynamics of a clamp-switch that recognizes a target DNA sequence through both Watson-Crick base pairing and triplex-forming Hoogsteen interactions. When compared to the equivalent linear DNA probe (which relies solely on Watson-Crick interactions), the extra Hoogsteen interactions in the DNA clamp-switch increase the probe's affinity for its target by ∼0.29 ± 0.02 kcal/mol/base. The Hoogsteen interactions of the clamp-switch likewise provide an additional specificity check that increases the discrimination efficiency toward a single-base mismatch by 1.2 ± 0.2 kcal/mol. This, in turn, leads to a 10-fold improvement in the width of the "specificity window" of this probe relative to that of the equivalent linear probe. Given these attributes, clamp-switches should be of utility not only for sensing applications but also, in the specific field of DNA nanotechnology, for applications calling for a better control over the building of nanostructures and nanomachines.

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

PubMed Central

Shih, Yan-Ping; Chou, Chi-Chi; Chen, Yi-Ling; Huang, Kai-Fa; Wang, Andrew H.- J.

2014-01-01

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities. PMID:24733552

Hyperpolarization-activated current (I(h)) in vestibular calyx terminals: characterization and role in shaping postsynaptic events.

PubMed

Meredith, Frances L; Benke, Tim A; Rennie, Katherine J

2012-12-01

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na(+) currents, voltage-dependent outward K(+) currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3-45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I(h)) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs(+) (1-5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I(h) was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I(h) was -123 mV, which shifted to -114 mV in the presence of cAMP. Activation of I(h) was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at -139 mV). Activation speeded up significantly (τ=136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I(h) could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I(h) was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin-Huxley type ionic conductances, removal of I(h) similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size

Relationship between glycaemic variability and hyperglycaemic clamp-derived functional variables in (impending) type 1 diabetes.

PubMed

Van Dalem, Annelien; Demeester, Simke; Balti, Eric V; Decochez, Katelijn; Weets, Ilse; Vandemeulebroucke, Evy; Van de Velde, Ursule; Walgraeve, An; Seret, Nicole; De Block, Christophe; Ruige, Johannes; Gillard, Pieter; Keymeulen, Bart; Pipeleers, Daniel G; Gorus, Frans K

2015-12-01

We examined whether measures of glycaemic variability (GV), assessed by continuous glucose monitoring (CGM) and self-monitoring of blood glucose (SMBG), can complement or replace measures of beta cell function and insulin action in detecting the progression of preclinical disease to type 1 diabetes. Twenty-two autoantibody-positive (autoAb(+)) first-degree relatives (FDRs) of patients with type 1 diabetes who were themselves at high 5-year risk (50%) for type 1 diabetes underwent CGM, a hyperglycaemic clamp test and OGTT, and were followed for up to 31 months. Clamp variables were used to estimate beta cell function (first-phase [AUC5-10 min] and second-phase [AUC120-150 min] C-peptide release) combined with insulin resistance (glucose disposal rate; M 120-150 min). Age-matched healthy volunteers (n = 20) and individuals with recent-onset type 1 diabetes (n = 9) served as control groups. In autoAb(+) FDRs, M 120-150 min below the 10th percentile (P10) of controls achieved 86% diagnostic efficiency in discriminating between normoglycaemic FDRs and individuals with (impending) dysglycaemia. M 120-150 min outperformed AUC5-10 min and AUC120-150 min C-peptide below P10 of controls, which were only 59-68% effective. Among GV variables, CGM above the reference range was better at detecting (impending) dysglycaemia than elevated SMBG (77-82% vs 73% efficiency). Combined CGM measures were equally efficient as M 120-150 min (86%). Daytime GV variables were inversely correlated with clamp variables, and more strongly with M 120-150 min than with AUC5-10 min or AUC120-150 min C-peptide. CGM-derived GV and the glucose disposal rate, reflecting both insulin secretion and action, outperformed SMBG and first- or second-phase AUC C-peptide in identifying FDRs with (impending) dysglycaemia or diabetes. Our results indicate the feasibility of developing minimally invasive CGM-based criteria for close metabolic monitoring and as outcome measures in trials.

MATLAB implementation of a dynamic clamp with bandwidth >125 KHz capable of generating INa at 37°C

PubMed Central

Clausen, Chris; Valiunas, Virginijus; Brink, Peter R.; Cohen, Ira S.

2012-01-01

We describe the construction of a dynamic clamp with bandwidth >125 KHz that utilizes a high performance, yet low cost, standard home/office PC interfaced with a high-speed (16 bit) data acquisition module. High bandwidth is achieved by exploiting recently available software advances (code-generation technology, optimized real-time kernel). Dynamic-clamp programs are constructed using Simulink, a visual programming language. Blocks for computation of membrane currents are written in the high-level matlab language; no programming in C is required. The instrument can be used in single- or dual-cell configurations, with the capability to modify programs while experiments are in progress. We describe an algorithm for computing the fast transient Na+ current (INa) in real time, and test its accuracy and stability using rate constants appropriate for 37°C. We then construct a program capable of supplying three currents to a cell preparation: INa, the hyperpolarizing-activated inward pacemaker current (If), and an inward-rectifier K+ current (IK1). The program corrects for the IR drop due to electrode current flow, and also records all voltages and currents. We tested this program on dual patch-clamped HEK293 cells where the dynamic clamp controls a current-clamp amplifier and a voltage-clamp amplifier controls membrane potential, and current-clamped HEK293 cells where the dynamic clamp produces spontaneous pacing behavior exhibiting Na+ spikes in otherwise passive cells. PMID:23224681

Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Mitchell; J Smith; M Mason

The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that ismore » directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.« less

Reversible fastener clamp load monitor with continuous visual or remote readout

NASA Astrophysics Data System (ADS)

Hodge, Malcolm H.; Kausel, Theodore C., Jr.; Begley, Matthew R.

1998-03-01

SIMS has developed a simple means for detecting and monitoring both absolute and relative clamp load, or bolt tension, in fastener systems. More than twenty-five percent of automotive failures are known to be due to undetected loss of fastener clamp load. While the equivalent aerospace maintenance statistics are not known, the average automobile has 3,500 fasteners while a Boeing 747 has closer to one million. It is therefore anticipated that the new SensaBolt clamp load tracking system could find wide applications in the aerospace arena. We describe a visually-evident and retrofitted clamp load monitoring design which is based on the differential joint substrate compression at, and immediately adjacent to, the fastener location. This intrinsically-accurate indicator does not necessarily require alteration in either the bolt or nut geometries, thereby facilitating product introduction and retrofit in aging aircraft applications. In addition, SensaBolt's sole reliance on substrate compression renders it more accurate then torque wrench or turn-of-nut techniques. Readout may be accomplished by any of three principal methods: for those applications with ease of access to the sensor, loss of tension can be determined by direct visual inspection. Application of a standard wrench can then be made to restore the fastener's proper tightness, per the SensaBolt indicators. In those instances where line-of-sight is unimpeded and more formal inspection is desired, the SensaBolt may be interrogated by a laser scanner bar code reader. Finally, SensaBolt may be addressed by the SIMS fiber optic harness for those instances where full-time remote interrogation is desired.

The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet. We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 “poremore » loops”, which then unfold MAD2 in the presence of ATP. N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.« less

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

PubMed Central

Landry, Aaron P.

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1. PMID:25147792

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

PubMed

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Relationship of dieting and restrained eating to self-reported caloric intake in female college freshmen.

PubMed

Goldstein, Stephanie P; Katterman, Shawn N; Lowe, Michael R

2013-04-01

Evidence indicates that restrained eaters do not eat less than unrestrained eaters in the natural environment. However, no study has examined caloric intake in those who are currently dieting to lose, or avoid gaining, weight. The current study examined caloric intake using 24-hour food recalls among individuals dieting to lose weight, dieting to avoid weight gain, restrained nondieters, and unrestrained nondieters. Participants were 246 female college students participating in a weight gain prevention trial. The predicted significant difference in caloric intake across the four groups was found for beverage but not for food intake. Results reinforce past literature indicating that dieting/restraint status does not reflect hypo-caloric intake in naturalistic settings. Copyright © 2012 Elsevier Ltd. All rights reserved.

Theoretical investigation of gain-clamped semiconductor optical amplifiers using the amplified spontaneous emission compensating effect

NASA Astrophysics Data System (ADS)

Jia, Xin-Hong

2006-12-01

The theoretical model on gain-clamped semiconductor optical amplifiers (GC-SOAs) based on compensating light has been constructed. Using this model, the effects of insertion position and peak reflectivity of the fiber Bragg grating (FBG) on the gain clamping and noise figure (NF) characteristics of GC-SOA are analyzed. The results show that the effect of the FBG insertion position on gain clamping is slight, but the lower NF can be obtained for input FBG-type GC-SOA; when the FBG peak wavelength is designed to close the signal wavelength, the gain clamping and NF characteristics that can be reached are better. Further study shows that, with the increased peak reflectivity of the FBG, the critical input power is broadened and the gain tends to be varied slowly; the larger bias current is helpful to raise gain and decrease the noise figure but is harmful to a gain flatness characteristic.

Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization

PubMed Central

Cadetti, Lucia; Bryson, Eric J.; Ciccone, Cory A.; Rabl, Katalin; Thoreson, Wallace B.

2008-01-01

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (−70 to −10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 μM) had no significant effect on rod calcium current (Ica) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 μM ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 μM) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods. PMID:16819987

Crystallized N-terminal domain of influenza virus matrix protein M1 and method of determining and using same

NASA Technical Reports Server (NTRS)

Luo, Ming (Inventor); Sha, Bingdong (Inventor)

2000-01-01

The matrix protein, M1, of influenza virus strain A/PR/8/34 has been purified from virions and crystallized. The crystals consist of a stable fragment (18 Kd) of the M1 protein. X-ray diffraction studies indicated that the crystals have a space group of P3.sub.t 21 or P3.sub.2 21. Vm calculations showed that there are two monomers in an asymmetric unit. A crystallized N-terminal domain of M1, wherein the N-terminal domain of M1 is crystallized such that the three dimensional structure of the crystallized N-terminal domain of M1 can be determined to a resolution of about 2.1 .ANG. or better, and wherein the three dimensional structure of the uncrystallized N-terminal domain of M1 cannot be determined to a resolution of about 2.1 .ANG. or better. A method of purifying M1 and a method of crystallizing M1. A method of using the three-dimensional crystal structure of M1 to screen for antiviral, influenza virus treating or preventing compounds. A method of using the three-dimensional crystal structure of M1 to screen for improved binding to or inhibition of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the manufacture of an inhibitor of influenza virus M1. The use of the three-dimensional crystal structure of the M1 protein of influenza virus in the screening of candidates for inhibition of influenza virus M1.

Crystal structure of the DNA polymerase III β subunit (β-clamp) from the extremophile Deinococcus radiodurans.

PubMed

Niiranen, Laila; Lian, Kjersti; Johnson, Kenneth A; Moe, Elin

2015-02-27

Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III β subunit (β-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans β-clamp (Drβ-clamp) at 2.0 Å resolution. The sequence verification process revealed that at the time of the study the gene encoding Drβ-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial β-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution. The structure of Drβ-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.

Disarming Batterers through Restraining Orders: The Promise and the Reality in California

ERIC Educational Resources Information Center

Seave, Paul L.

2006-01-01

Laws that prohibit persons under a domestic violence restraining order from purchasing or possessing a firearm are a primary way to keep guns out of the hands of batterers. In July 2005, the California Attorney General's Task Force on the Local Criminal Justice Response to Domestic Violence issued a report called Keeping the Promise: Victim Safety…

Emotional arousal and overeating in restrained eaters.

PubMed

Cools, J; Schotte, D E; McNally, R J

1992-05-01

We tested the effects of 3 mood inductions (neutral, positive, and negative) on food intake in 91 women of varying degrees of dietary restraint. Mood induction was accomplished by exposure to 1 of 3 film segments: a travelogue (neutral affect), a comedy film (positive affect), and a horror film (negative affect). In subjects exposed to the neutral film, food intake decreased with increasing levels of dietary restraint. Among subjects who viewed either the comedy film or the horror film, however, food intake increased with increasing restraint. Although the horror film appeared to be more disinhibiting than the comedy film, this effect may have resulted from a difference in the intensity of the emotions induced rather than from their valence. These results suggest that emotional arousal, regardless of valence, may trigger overeating among restrained eaters.

Removing guns from batterers: findings from a pilot survey of domestic violence restraining order recipients in California.

PubMed

Vittes, Katherine A; Webster, Daniel W; Frattaroli, Shannon; Claire, Barbara E; Wintemute, Garen J

2013-05-01

Persons under certain domestic violence restraining orders in California are required to surrender any firearms in their possession within 24 hours of service. The California Department of Justice funded a pilot program in which Sheriff's Offices in two counties developed a system for better enforcing the firearm surrender requirement. As part of a larger process evaluation, 17 restraining order recipients were interviewed about their experiences with and feelings about the removal of firearms from their abusers. Most women surveyed wanted firearms removed and felt safer as a result of their removal. Implications of the findings are discussed.

Adaptively restrained molecular dynamics in LAMMPS

NASA Astrophysics Data System (ADS)

Kant Singh, Krishna; Redon, Stephane

2017-07-01

Adaptively restrained molecular dynamics (ARMD) is a recently introduced particles simulation method that switches positional degrees of freedom on and off during simulation in order to speed up calculations. In the NVE ensemble, ARMD allows users to trade between precision and speed while, in the NVT ensemble, it makes it possible to compute statistical averages faster. Despite the conceptual simplicity of the approach, however, integrating it in existing molecular dynamics packages is non-trivial, in particular since implemented potentials should a priori be rewritten to take advantage of frozen particles and achieve a speed-up. In this paper, we present novel algorithms for integrating ARMD in LAMMPS, a popular multi-purpose molecular simulation package. In particular, we demonstrate how to enable ARMD in LAMMPS without having to re-implement all available force fields. The proposed algorithms are assessed on four different benchmarks, and show how they allow us to speed up simulations up to one order of magnitude.

Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases

PubMed Central

Lamb, Brian M.; Mercer, Andrew C.; Barbas, Carlos F.

2013-01-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5′-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5′ T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5′ T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes. PMID:23980031

Structure of the N-terminal fragment of Escherichia coli Lon protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Mi; Gustchina, Alla; Rasulova, Fatima S.

2010-10-22

The structure of a recombinant construct consisting of residues 1-245 of Escherichia coli Lon protease, the prototypical member of the A-type Lon family, is reported. This construct encompasses all or most of the N-terminal domain of the enzyme. The structure was solved by SeMet SAD to 2.6 {angstrom} resolution utilizing trigonal crystals that contained one molecule in the asymmetric unit. The molecule consists of two compact subdomains and a very long C-terminal {alpha}-helix. The structure of the first subdomain (residues 1-117), which consists mostly of {beta}-strands, is similar to that of the shorter fragment previously expressed and crystallized, whereas themore » second subdomain is almost entirely helical. The fold and spatial relationship of the two subdomains, with the exception of the C-terminal helix, closely resemble the structure of BPP1347, a 203-amino-acid protein of unknown function from Bordetella parapertussis, and more distantly several other proteins. It was not possible to refine the structure to satisfactory convergence; however, since almost all of the Se atoms could be located on the basis of their anomalous scattering the correctness of the overall structure is not in question. The structure reported here was also compared with the structures of the putative substrate-binding domains of several proteins, showing topological similarities that should help in defining the binding sites used by Lon substrates.« less

Glomerular loss after arteriovenous and arterial clamping for renal warm ischemia in a swine model.

PubMed

Bechara, Gustavo Ruschi; Damasceno-Ferreira, José Aurelino; Abreu, Leonardo Albuquerque Dos Santos; Costa, Waldemar Silva; Sampaio, Francisco José Barcellos; Pereira-Sampaio, Marco Aurélio; Souza, Diogo Benchimol De

2016-11-01

To evaluate the glomerular loss after arteriovenous or arterial warm ischemia in a swine model. Twenty four pigs were divided into Group Sham (submitted to all surgical steps except the renal ischemia), Group AV (submitted to 30 minutes of warm ischemia by arteriovenous clamping of left kidney vessels), and Group A (submitted to 30 minutes of ischemia by arterial clamping). Right kidneys were used as controls. Weigh, volume, cortical volume, glomerular volumetric density (Vv[Glom]), volume-weighted glomerular volume (VWGV), and the total number of glomeruli were measured for each organ. Group AV showed a 24.5% reduction in its left kidney Vv[Glom] and a 25.4% reduction in the VWGV, when compared to the right kidney. Reductions were also observed when compared to kidneys of sham group. There was a reduction of 19.2% in the total number of glomeruli in AV kidneys. No difference was observed in any parameters analyzed on the left kidneys from group A. Renal warm ischemia of 30 minutes by arterial clamping did not caused significant glomerular damage, but arteriovenous clamping caused significant glomerular loss in a swine model. Clamping only the renal artery should be considered to minimize renal injury after partial nephrectomies.

The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

PubMed

Taki, Masumi; Kuroiwa, Hiroyuki; Sisido, Masahiko

2009-01-01

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

PubMed

Polyansky, Anton A; Vassilevski, Alexander A; Volynsky, Pavel E; Vorontsova, Olga V; Samsonova, Olga V; Egorova, Natalya S; Krylov, Nicolay A; Feofanov, Alexei V; Arseniev, Alexander S; Grishin, Eugene V; Efremov, Roman G

2009-07-21

In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin

PubMed Central

Skotland, Tore; Holm, Turid; Østerud, Bjarne; Flengsrud, Ragnar; Prydz, Hans

1974-01-01

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000±2000 in sodium dodecyl sulphate–polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca2+ and is adsorbed to BaSO4. The pKa of its BaSO4-binding group(s) is 3.1–3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO4 and, since the ability of the whole protein to bind to BaSO4 is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid. ImagesFig. 2. PMID:4219283

A self-strain feedback tuning-fork-shaped ionic polymer metal composite clamping actuator with soft matter elasticity-detecting capability for biomedical applications.

PubMed

Feng, Guo-Hua; Huang, Wei-Lun

2014-12-01

This paper presents a smart tuning-fork-shaped ionic polymer metal composite (IPMC) clamping actuator for biomedical applications. The two fingers of the actuator, which perform the clamping motion, can be electrically controlled through a unique electrode design on the IPMC material. The generated displacement or strain of the fingers can be sensed using an integrated soft strain-gage sensor. The IPMC actuator and associated soft strain gage were fabricated using a micromachining technique. A 13.5×4×2 mm(3) actuator was shaped from Nafion solution and a selectively grown metal electrode formed the active region. The strain gage consisted of patterned copper foil and polyethylene as a substrate. The relationship between the strain gage voltage output and the displacement at the front end of the actuator's fingers was characterized. The equivalent Young's modulus, 13.65 MPa, of the soft-strain-gage-integrated IPMC finger was analyzed. The produced clamping force exhibited a linear increasing rate of 1.07 mN/s, based on a dc driving voltage of 7 V. Using the developed actuator to clamp soft matter and simultaneously acquire its Young's modulus was achieved. This demonstrated the feasibility of the palpation function and the potential use of the actuator in minimally invasive surgery. Copyright © 2014 Elsevier B.V. All rights reserved.

Modulation by clamping: Kv4 and KChIP interactions.

PubMed

Wang, Kewei

2008-10-01

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) alpha subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K+ current, I(SA), in neurons and transient outward current, I(TO), in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

PubMed Central

Ren, Xiao-Ming; Li, De-Feng; Jiang, Shuai; Lan, Xian-Qing; Hu, Yonglin; Sun, Hui; Wang, Da-Cheng

2015-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently. PMID:26114302

Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal

PubMed Central

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF), N-terminal sequence (219 bp, ΔespFN), and C-terminal sequence (528 bp, ΔespFC) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN, and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), ΔespF, ΔespF/pespF, ΔespFC, ΔespFC/pespFC, ΔespFN, and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN, ΔespFC/pespFC, and ΔespFN/pespFN group (p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain. PMID:28983470

Autoantibodies to N-terminally truncated GAD improve clinical phenotyping of individuals with adult-onset diabetes: Action LADA 12.

PubMed

Achenbach, Peter; Hawa, Mohammed I; Krause, Stephanie; Lampasona, Vito; Jerram, Samuel T; Williams, Alistair J K; Bonifacio, Ezio; Ziegler, Anette G; Leslie, R David

2018-07-01

Adult-onset type 1 diabetes, in which the 65 kDa isoform of GAD (GAD65) is a major autoantigen, has a broad clinical phenotype encompassing variable need for insulin therapy. This study aimed to evaluate whether autoantibodies against N-terminally truncated GAD65 more closely defined a type 1 diabetes phenotype associated with insulin therapy. Of 1114 participants with adult-onset diabetes from the Action LADA (latent autoimmune diabetes in adults) study with sufficient sera, we selected those designated type 1 (n = 511) or type 2 diabetes (n = 603) and retested the samples in radiobinding assays for human full-length GAD65 autoantibodies (f-GADA) and N-terminally truncated (amino acids 96-585) GAD65 autoantibodies (t-GADA). Individuals' clinical phenotypes were analysed according to antibody binding patterns. Overall, 478 individuals were f-GADA-positive, 431 were t-GADA-positive and 628 were negative in both assays. Risk of insulin treatment was augmented in t-GADA-positive individuals (OR 4.69 [95% CI 3.57, 6.17]) compared with f-GADA-positive individuals (OR 3.86 [95% CI 2.95, 5.06]), irrespective of diabetes duration. Of 55 individuals who were f-GADA-positive but t-GADA-negative, i.e. with antibody binding restricted to the N-terminus of GAD65, the phenotype was similar to type 2 diabetes with low risk of progression to insulin treatment. Compared with these individuals with N-terminal GAD65-restricted GADA, t-GADA-positive individuals were younger at diagnosis (p = 0.005), leaner (p < 0.0001) and more often had multiple diabetes-associated autoantibodies (28.3% vs 7.3%; p = 0.0005). In individuals with adult-onset diabetes, presence of N-terminally truncated GAD65 autoantibodies is associated with the clinical phenotype of autoimmune type 1 diabetes and predicts insulin therapy.

[Focus on placental transfusion for preterm neonates: Delayed cord clamping and/or milking?

PubMed

Sorin, G; Tosello, B

2016-11-01

Anemia of prematurity remains a common complication despite recent advances in perinatal and neonatal medicine. The delayed cord clamping (at least 30seconds as recommended) has several benefits: increased hemoglobin and hematocrit levels at birth, improved initial hemodynamic, decreased incidence of transfusions and intraventricular hemorrhages. When the birth transition is difficult, wait 30seconds before clamping can be impossible. So as not to interfere with the neonatal resuscitation, the "milking" has been proposed as an alternative method to the delayed cord clamping. This is a safe and easy method, which can be done either by an obstetrician or pediatrician with comparable results for the child on his hemodynamic, hematological and neurological status. It still lacks technical information on this method and neurodevelopmental outcomes of these preterm infants. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

[Correlation between thermostability of the xylanase EvXyn11(TS) and its N-terminal disulfide bridge].

PubMed

Min, Rou; Li, Jianfang; Gao, Shujuan; Zhang, Huimin; Wu, Jing; Wu, Minchen

2013-04-04

To reveal the correlation between thermostability of xylanase EvXyn11(TS) and its N-terminal disulfide bridge, an EvXyn11(TS)-encoding gene (Syxyn11) was synthesized and subjected to site-directed mutagenesis. Multiple homology alignment of protein primary structures between the EvXyn11(TS) and several GH family 11 xylanases displayed that, in their N-termini, only EvXyn11(TS) contained a disulfide bridge (Cys5-Cys32), whose effect on the xylanase thermostability was predicted by molecular dynamics simulation. We constructed a gene Syxyn11(M), encoding the mutated xylanase (EvXyn11(M)) without N-terminal disulfide bridge. Then, Syxyn11 and Syxyn11(M) were expressed in Pichia pastoris GS115, and temperature and pH properties of the expressed enzymes were analyzed. The analytical results displayed that the temperature optimum of EvXyn11(M) was 70 degrees C, which was 15 degrees C lower than that of EvXyn11(TS). The half-life (t1/2(90)) of EvXyn11(TS) at 90 degrees C was 32 min, while the t1/2(70) of EvXyn11(M) at 70 degrees C was only 8.0 min. The important role of the N-terminal disulfide bridge on the thermostability of EvXyn11(TS) was first predicted by molecular dynamics simulation, and confirmed by site-directed mutagenesis. This work provided a novel strategy to improve thermostabilities of the mesophilic family 11 xylanases with high specific activities.

A nonlinear theory for spinning anisotropic beams using restrained warping functions

NASA Technical Reports Server (NTRS)

Ie, C. A.; Kosmatka, J. B.

1993-01-01

A geometrically nonlinear theory is developed for spinning anisotropic beams having arbitrary cross sections. An assumed displacement field is developed using the standard 3D kinematics relations to describe the global beam behavior supplemented with an additional field that represents the local deformation within the cross section and warping out of the cross section plane. It is assumed that the magnitude of this additional field is directly proportional to the local stress resultants. In order to take into account the effects of boundary conditions, a restraining function is introduced. This function plays the role of reducing the amount of free warping deformation throughout the field due to the restraint of the cross section(s) at the end(s) of the beam, e.g., in the case of a cantilever beam. Using a developed ordering scheme, the nonlinear strains are calculated to the third order. The FEM is developed using the weak form variational formulation. Preliminary interesting numerical results have been obtained that indicate the role of the restraining function in the case of a cantilever beam with circular cross section. These results are for the cases of a tip displacement (static) and free vibration studies for both isotropic and anisotropic materials with varied fiber orientations.

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration.

PubMed

Ibrahim, Hisham R; Imazato, Kenta; Ono, Hajime

2011-09-28

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.

Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

PubMed

Traverso, José A; Micalella, Chiara; Martinez, Aude; Brown, Spencer C; Satiat-Jeunemaître, Béatrice; Meinnel, Thierry; Giglione, Carmela

2013-03-01

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

Clamped seismic metamaterials: ultra-low frequency stop bands

NASA Astrophysics Data System (ADS)

Achaoui, Y.; Antonakakis, T.; Brûlé, S.; Craster, R. V.; Enoch, S.; Guenneau, S.

2017-06-01

The regularity of earthquakes, their destructive power, and the nuisance of ground vibration in urban environments, all motivate designs of defence structures to lessen the impact of seismic and ground vibration waves on buildings. Low frequency waves, in the range 1-10 Hz for earthquakes and up to a few tens of Hz for vibrations generated by human activities, cause a large amount of damage, or inconvenience; depending on the geological conditions they can travel considerable distances and may match the resonant fundamental frequency of buildings. The ultimate aim of any seismic metamaterial, or any other seismic shield, is to protect over this entire range of frequencies; the long wavelengths involved, and low frequency, have meant this has been unachievable to date. Notably this is scalable and the effects also hold for smaller devices in ultrasonics. There are three approaches to obtaining shielding effects: bragg scattering, locally resonant sub-wavelength inclusions and zero-frequency stop-band media. The former two have been explored, but the latter has not and is examined here. Elastic flexural waves, applicable in the mechanical vibrations of thin elastic plates, can be designed to have a broad zero-frequency stop-band using a periodic array of very small clamped circles. Inspired by this experimental and theoretical observation, all be it in a situation far removed from seismic waves, we demonstrate that it is possible to achieve elastic surface (Rayleigh) wave reflectors at very large wavelengths in structured soils modelled as a fully elastic layer periodically clamped to bedrock. We identify zero frequency stop-bands that only exist in the limit of columns of concrete clamped at their base to the bedrock. In a realistic configuration of a sedimentary basin 15 m deep we observe a zero frequency stop-band covering a broad frequency range of 0-30 Hz.

Small Molecules for Early Endosome-Specific Patch Clamping.

PubMed

Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering a thermostable fungal GH10 xylanase, importance of N-terminal amino acids.

PubMed

Song, Letian; Tsang, Adrian; Sylvestre, Michel

2015-06-01

Xylanases are used in many industrial processes including pulp bleaching, baking, detergent, and the hydrolysis of plant cell wall in biofuels production. In this work we have evolved a single domain GH10 xylanase, Xyn10A_ASPNG, from Aspergillus niger to improve its thermostability. We introduced a rational approach involving as the first step a computational analysis to guide the design of a mutagenesis library in targeted regions which identified thermal important residues that were subsequently randomly mutagenized through rounds of iterative saturation mutagenesis (ISM). Focusing on five residues, four rounds of ISM had generated a quintuple mutant 4S1 (R25W/V29A/I31L/L43F/T58I) which exhibited thermal inactivation half-life (t1/2 ) at 60°C that was prolonged by 30 folds in comparison with wild-type enzyme. Whereas the wild-type enzyme retained 0.2% of its initial activity after a heat treatment of 10 min at 60°C and was completely inactivated after 2 min at 65°C, 4S1 mutant retained 30% of its initial activity after 15 min heating at 65°C. Furthermore, the mutant melting temperature (Tm ) increased by 17.4°C compared to the wild type. Each of the five mutations in 4S1 was found to contribute to thermoresistance, but the dramatic improvement of enzyme thermoresistance of 4S1 was attributed to the synergistic effects of the five mutations. Comparison of biochemical data and model structure between 4S1 and the wild-type enzyme suggested that the N-terminal coil of the enzyme is important in stabilizing GH10 xylanase structure. Based on model structure analyses, we propose that enforced hydrophobic interactions within N-terminal elements and between N- and C-terminal ends are responsible for the improved thermostability of Xyn10A_ASPNG. © 2015 Wiley Periodicals, Inc.

N-terminal acetylation -an Essential Protein Modification Emerges as an Important Regulator of Stress Responses.

PubMed

Linster, Eric; Wirtz, Markus

2018-06-26

N-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. The majority of proteins is acetylated at their N-terminus in a co-translational manner by ribosome-associated N-terminal acetyltransferases (NAT). However, the recent discovery of Golgi-membrane localized NATs in metazoan, and plastid-localized NATs in plants challenged the dogma of static, co-translational imprinting of the proteome by NTA. Indeed, NTA by the cytosolic NatA is highly dynamic and under hormonal control in plants. Such active control has not been evidenced yet in other eukaryotes and might be an adaptation to the sessile lifestyle of plants forcing them to cope with diverse environmental challenges. The function of NTA for individual proteins is distinct and yet unpredictable. In yeast and humans, NTA has been shown to affect protein-protein interactions, subcellular localization, folding, aggregation, or degradation of a handful of proteins. In particular, the impact of NTA on the protein-turnover is documented by diverse examples in yeast. Consequently, NTA has recently dicovered to be a degradation signal in a distinct branch of the N-end rule pathway ubiquitin-mediated proteolysis. In this review, we summarize the current knowledge on the NAT machinery in higher plants and discuss the potential function of NTA during biotic and abiotic stresses.

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

PubMed

Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

2012-03-29

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN. Published by Elsevier Ltd.

The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

PubMed Central

Ampah-Korsah, Henry; Anderberg, Hanna I.; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

2016-01-01

Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

Sequential N-Terminal Pro-B-Type Natriuretic Peptide and High-Sensitivity Cardiac Troponin Measurements During Albumin Replacement in Patients With Severe Sepsis or Septic Shock.

PubMed

Masson, Serge; Caironi, Pietro; Fanizza, Caterina; Carrer, Sara; Caricato, Anselmo; Fassini, Paola; Vago, Tarcisio; Romero, Marilena; Tognoni, Gianni; Gattinoni, Luciano; Latini, Roberto

2016-04-01

Myocardial dysfunction is a frequent complication in patients with severe sepsis and can worsen the prognosis. We investigated whether circulating biomarkers related to myocardial function and injury predicted outcome and were associated with albumin replacement. A multicenter, randomized clinical trial about albumin replacement in severe sepsis or septic shock (the Albumin Italian Outcome Sepsis trial). Forty ICUs in Italy. Nine hundred and ninety-five patients with severe sepsis or septic shock. Randomization to albumin and crystalloid solutions or crystalloid solutions alone. Plasma concentrations of N- terminal pro-B-type natriuretic peptide and high-sensitivity cardiac troponin T were measured 1, 2, and 7 days after enrollment. We tested the relationship of single marker measurements or changes over time with clinical events, organ dysfunctions, albumin replacement, and ICU or 90-day mortality in the overall population and after stratification by shock. N-terminal pro-B-type natriuretic peptide levels were abnormal in 97.4% of the patients and high-sensitivity cardiac troponin T in 84.5%, with higher concentrations in those with shock. After extensive adjustments, N-terminal pro-B-type natriuretic peptide concentrations predicted ICU or 90-day mortality, better than high-sensitivity cardiac troponin T. Early changes in N-terminal pro-B-type natriuretic peptide or high-sensitivity cardiac troponin T concentrations were independently associated with subsequent mortality in patients with shock. Patients given albumin had significantly higher N-terminal pro-B-type natriuretic peptide levels; in addition, early rise in N-terminal pro-B-type natriuretic peptide was associated with a better outcome in this subgroup. Circulating N-terminal pro-B-type natriuretic peptide and high-sensitivity cardiac troponin T are frequently elevated in severe sepsis or septic shock and have relevant prognostic value, which may be important in monitoring the clinical efficacy of

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

PubMed Central

Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

2015-01-01

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

Seismic rehabilitation of skewed and curved bridges using a new generation of buckling restrained braces.

DOT National Transportation Integrated Search

2016-12-01

The objective of this project is to find effective configurations for using buckling restrained braces (BRBs) in both skewed and curved bridges for reducing the effects of strong earthquakes. Verification is performed by numerical simulation using an...

Robotic multi-well planar patch-clamp for native and primary mammalian cells

PubMed Central

Milligan, Carol J; Li, Jing; Sukumar, Piruthivi; Majeed, Yasser; Dallas, Mark L; English, Anne; Emery, Paul; Porter, Karen E; Smith, Andrew M; McFadzean, Ian; Beccano-Kelly, Dayne; Bahnasi, Yahya; Cheong, Alex; Naylor, Jacqueline; Zeng, Fanning; Liu, Xing; Gamper, Nikita; Jiang, Lin-Hua; Pearson, Hugh A; Peers, Chris; Robertson, Brian; Beech, David J

2009-01-01

Multi-well robotic planar patch-clamp has become common in drug development and safety programmes because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favoured method. Here we show the wider potential of the multi-well approach with the capability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints, and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by pre-programmed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 hr depending on the experimental design and yields 16-33 cell recordings. PMID:19197268

Clamp-crushing vs. radiofrequency-assisted liver resection:changes in liver function tests.

PubMed

Palibrk, Ivan; Milicic, Biljana; Stojiljkovic, Ljuba; Manojlovic, Nebojsa; Dugalic, Vladimir; Bumbasirevic, Vesna; Kalezic, Nevena; Zuvela, Marinko; Milicevic, Miroslav

2012-05-01

Liver resection is the gold standard in managing patients with metastatic or primary liver cancer. The aim of our study was to compare the traditional clamp-crushing technique to the radiofrequency- assisted liver resection technique in terms of postoperative liver function. Liver function was evaluated preoperatively and on postoperative days 3 and 7. Liver synthetic function parameters (serum albumin level, prothrombin time and international normalized ratio), markers of hepatic injury and necrosis (serum alanine aminotransferase, aspartate aminotransferase and total bilirubin level) and microsomal activity (quantitative lidocaine test) were compared. Forty three patients completed the study (14 had clamp-crushing and 29 had radiofrequency assisted liver resection). The groups did not differ in demographic characteristics, pre-operative liver function, operative time and perioperative transfusion rate. In postoperative period, there were similar changes in monitored parameters in both groups except albumin levels, that were higher in radiofrequency-assisted liver resection group (p=0.047). Both, traditional clamp-crushing technique and radiofrequency assisted liver resection technique, result in similar postoperative changes of most monitored liver function parameters.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

PubMed Central

Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

2010-01-01

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

Safety of minimally invasive mitral valve surgery without aortic cross-clamp.

PubMed

Umakanthan, Ramanan; Leacche, Marzia; Petracek, Michael R; Kumar, Sathappan; Solenkova, Nataliya V; Kaiser, Clayton A; Greelish, James P; Balaguer, Jorge M; Ahmad, Rashid M; Ball, Stephen K; Hoff, Steven J; Absi, Tarek S; Kim, Betty S; Byrne, John G

2008-05-01

We developed a technique for open heart surgery through a small (5 cm) right-anterolateral thoracotomy without aortic cross-clamp. One hundred and ninety-five consecutive patients (103 male and 92 female), age 69 +/- 8 years, underwent surgery between January 2006 and July 2007. Mean preoperative New York Heart Association function class was 2.2 +/- 0.7. Thirty-five patients (18%) had an ejection fraction 0.35 or less. Cardiopulmonary bypass was instituted through femoral (176 of 195, 90%), axillary (18 of 195, 9%), or direct aortic (1 of 195, 0.5%) cannulation. Under cold fibrillatory arrest (mean temperature 28.2 degrees C) without aortic cross-clamp, mitral valve repair (72 of 195, 37%), mitral valve replacement (117 of 195, 60%), or other (6 of 195, 3%) procedures were performed. Concomitant procedures included maze (45 of 195, 23%), patent foramen ovale closure (42 of 195, 22%) and tricuspid valve repair (16 of 195, 8%), or replacement (4 of 195, 2%). Thirty-day mortality was 3% (6 of 195). Duration of fibrillatory arrest, cardiopulmonary bypass, and "skin to skin" surgery were 88 +/- 32, 118 +/- 52, and 280 +/- 78 minutes, respectively. Ten patients (5%) underwent reexploration for bleeding and 44% did not receive any blood transfusions. Six patients (3%) sustained a postoperative stroke, eight (4%) developed low cardiac output syndrome, and two (1%) developed renal failure requiring hemodialysis. Mean length of hospital stay was 7 +/- 4.8 days. This simplified technique of minimally invasive open heart surgery is safe and easily reproducible. Fibrillatory arrest without aortic cross-clamping, with coronary perfusion against an intact aortic valve, does not increase the risk of stroke or low cardiac output. It may be particularly useful in higher risk patients in whom sternotomy with aortic clamping is less desirable.

Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.

PubMed

Hoersch, Daniel; Otto, Harald; Cusanovich, Michael A; Heyn, Maarten P

2009-07-14

The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is approximately 20 A away from the chromophore binding pocket and separated from it by the central beta-sheet. The question of the propagation of the structural change across the central beta-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix alpha1 near the functionally important salt bridge E12-K110 between the N-terminal cap and the central anti-parallel beta-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I(2) intermediate, which is formed in approximately 200 mus, the change near N13 is delayed and rises with the next intermediate I(2)', which forms in approximately 2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central beta-sheet via the N-terminal region to helix alpha1

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

PubMed Central

Song, Albert S.; Poor, Taylor A.; Abriata, Luciano A.; Jardetzky, Theodore S.; Dal Peraro, Matteo; Lamb, Robert A.

2016-01-01

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin–neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design. PMID:27335462

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins.

PubMed

Song, Albert S; Poor, Taylor A; Abriata, Luciano A; Jardetzky, Theodore S; Dal Peraro, Matteo; Lamb, Robert A

2016-07-05

Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin-neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

PubMed

Tanco, Sebastian; Aviles, Francesc Xavier; Gevaert, Kris; Lorenzo, Julia; Van Damme, Petra

2017-01-01

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

Oxidation-specific epitopes restrain bone formation.

PubMed

Ambrogini, Elena; Que, Xuchu; Wang, Shuling; Yamaguchi, Fumihiro; Weinstein, Robert S; Tsimikas, Sotirios; Manolagas, Stavros C; Witztum, Joseph L; Jilka, Robert L

2018-06-06

Atherosclerosis and osteoporosis are epidemiologically linked and oxidation specific epitopes (OSEs), such as phosphocholine (PC) of oxidized phospholipids (PC-OxPL) and malondialdehyde (MDA), are pathogenic in both. The proatherogenic effects of OSEs are opposed by innate immune antibodies. Here we show that high-fat diet (HFD)-induced bone loss is attenuated in mice expressing a single chain variable region fragment of the IgM E06 (E06-scFv) that neutralizes PC-OxPL, by increasing osteoblast number and stimulating bone formation. Similarly, HFD-induced bone loss is attenuated in mice expressing IK17-scFv, which neutralizes MDA. Notably, E06-scFv also increases bone mass in mice fed a normal diet. Moreover, the levels of anti-PC IgM decrease in aged mice. We conclude that OSEs, whether produced chronically or increased by HFD, restrain bone formation, and that diminished defense against OSEs may contribute to age-related bone loss. Anti-OSEs, therefore, may represent a novel therapeutic approach against osteoporosis and atherosclerosis simultaneously.

Which aortic clamp strategy is better to reduce postoperative stroke and death

PubMed Central

Chen, Liyu; Hua, Xiumeng; Song, Jiangping; Wang, Liqing

2018-01-01

Abstract Background: Stroke is severe complication of coronary artery bypass grafting (CABG) which may be associated with clamp strategy, there are 2 strategies to clamp aorta including single aortic clamp (SAC) and partial aortic clamp (PAC). It is controversial that which clamping strategy is better to reduce the postoperative stroke and death, so this study aims to investigate which is better for reducing postoperative stroke and death within 30 days. Methods: We collected 469 patients who had on-pump CABG in Fuwai Hospital during January 2014 to July 2015. The SAC group consisted of 265 patients while the PAC group included 204 patients. We compared the 2 group patient difference. At the same time, 12 studies were identified by systematic search. The odds ratio (OR) was used as effect index to compare SAC and PAC strategy by fix-effect modeling. We also tested heterogeneity and publication bias. The primary end point of study was occurrence of postoperative stroke within 30 days of operation, the second end point of study was the incidence of 30-day mortality. Results: The single center retrospective study showed that the patients in the SAC group were older than those in the PAC group (62.5 ± 8.1 vs 60.3 ± 8.0 years, P = .01). The proportions of peripheral vascular disease and hypertension of SAC were higher than PAC (71 (26.8%) versus 36 (17.6%), P = .02; 183 (69.1%) versus 115 (56.4%), P = .01, respectively). Besides, the number of vascular anastomosis was more in the SAC group (3.29 ± 0.74 versus 2.97 ± 0.974, P < .001). The linear-regression analysis suggested that the time of cardiopulmonary bypass of SAC was shorter than the PAC group (93.2 ± 22.4 vs.103.4 ± 26.8 minutes, P-regression < .001) and postoperative death within 30-days was similar (1 (0.4%) vs. 2 (1.0%), P-regression = .47). There was no stroke occurring in both the groups. And the meta-analysis suggested the postoperative stroke and

Enhanced strain effect of aged acceptor-doped BaTiO3 ceramics with clamping domain structures

NASA Astrophysics Data System (ADS)

Wang, Lei; Zhou, Zhiyong; Zhao, Xiaobo; Liu, Zhen; Liang, Ruihong; Dong, Xianlin

2017-03-01

A clamping domain structure is proposed to improve the amount of non-180° domain switching in BaTiO3 based piezoelectric ceramics. Experimental results show a large unipolar strain of 0.23% at 5 kV/mm in aged 0.5 mol. % Mn doped BaTiO3 ceramics with clamping domain structures, and the normalized strain (d33*= Smax/Emax) reaches 600 pm/V at low electric fields of 2 or 3 kV/mm. In contrast, pure BaTiO3 ceramics with clamping domain structures exhibit no clear polarization constriction or strain enhancement at 3 kV/mm. Electron paramagnetic resonance spectra verify the existence of titanium vacancies, Mn2+ and Mn4+, in 0.5 mol. % Mn doped BaTiO3 ceramics. These results indicate that the enhanced strain effect can be attributed to the combined effect of the clamping domain structure and stabilization of defect dipoles. This method provides a general way to obtain large strain in ferroelectrics.