bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal Amyloidosis in Dogs: A Retrospective Study of 91 Cases with Comparison of the Disease betweenShar‐Pei and Non‐Shar‐Pei Dogs
Authors: Segev G. , Cowgill L. D. , Jessen S. , Berkowitz A. , Mohr C.F. , Aroch I.
Published: January 2012

Background

Renal amyloidosis (RA) is a progressive and fatal renal disease.

Hypothesis

Clinical and pathologic manifestations ofRAdiffer betweenChineseShar‐Pei (CSPs) and non‐Shar‐Pei (NSPs) dogs.

Animals

91 client‐owned dogs.

Methods

Retrospective review of medical records of dogs with a histological diagnosis ofRA. Clinical and clinicopathologic data, hospitalization, complications, and outcome were compared betweenCSPs andNSPs.

Results

Comorbid diseases were present in 64% of all dogs.CSPs were significantly younger compared toNSPs (median, 4.8 years; range: 3.6–17 versus median: 9.0 years; range: 2.4–11.1;P < .0001). The frequency of hypoalbuminemia, the most common biochemical abnormality, was higher inNSPs compared to CSPs (100% versus 64.7%, respectively;P < .001). Median serum creatinine concentration at presentation was 5.5 mg/dL, and was 3‐fold higher inCSPs compared toNSPs (P = .005). Increased urine protein : creatinine ratio was present in 96% of all dogs. Nephrotic syndrome was present in 10% ofNSPs but not inCSPs. Glomerular amyloid deposition, present in bothCSPs (78.6%) andNSPs (95.6%) was most commonly diffuse, global, and severe. Renal medullar amyloidosis was more common inCSPs (100%) compared to NSPs (49.0%,P = .002), as was extrarenal amyloid deposition. The median survival time of all dogs was 5 days (range: 0–443 days). Serum creatinine concentration was significantly and negatively associated with survival (P = .025).

Conclusions and Clinical Relevance

The clinical and pathologic manifestations of amyloidosis differ betweenCSPs andNSPs. The survival time observed herein was unexpectedly low, and argues for early surveillance and management of the underlying predisposing conditions.
Comparison ofIVandIMFormulations of SyntheticACTHforACTHStimulation Tests in Healthy Dogs
Authors: Cohen T.A. , Feldman E.C.
Published: January 2012

Background

Two commercially available forms of syntheticACTHare used to diagnose and monitor hyper‐ and hypoadrenocorticism in dogs.

Objective

To compare the biologic activity of the liquid and lyophilized forms of cosyntropin.

Animals

Eighteen privately owned healthy dogs were included.

Methods

Dogs were assigned to one of 2 groups of 9 dogs each. Group 1 dogs were tested with the lyophilized product first and the liquid solution 30–60 days later. The Group 2 dogs were tested with the liquid solution first and the lyophilized drug 30–60 days later. For theACTHstimulation tests, serum samples were collected before and 1 hour afterIMadministration of 0.25 mg reconstituted lyophilized product or 1 hour afterIVadministration of 0.25 mg of liquid solution. Cortisol concentrations of all serum samples were measured by use of a commercial cortisol radioimmunoassay.

Results

Serum cortisol concentrations before and afterACTHstimulation did not differ significantly between groups (P = .57). In addition, no individual dog had as much as a 20% difference in serum cortisol concentrations after administration of eitherACTHformulation.

Conclusions and Clinical Importance

Given the lack of significant differences of theACTHstimulation test results, the lyophilized and liquid solution products can be used interchangeably.
The role of movement exaggeration in the anticipation of deceptive soccer penalty kicks
Authors: Smeeton N.J. , Williams A.M.
Published: January 2012

Human movement containing deception about the true outcome is thought to be perceived differently compared to the non‐deceptive version. Exaggeration in the movement is thought to change the perceiver's mode of functioning from an invariant to a cue‐based mode. We tested these ideas by examining anticipation in skilled and less skilled soccer players while they viewed temporally occluded (−240 ms, −160 ms, −80 ms, 0 ms, +80 ms) deceptive, non‐deceptive, and non‐deceptive‐exaggerated penalty kicks. Kinematic analyses were used to ascertain that the kicking actions differed across conditions. The accuracy of judging the direction of an opponent's kick as well as response confidence were recorded. Players were over confident when anticipating deceptive penalty kicks compared to non‐deceptive kicks, suggesting a cue‐based mode was used. Furthermore, there was a significant relationship between less skilled players’ confidence ratings and their accuracy 80 ms before ball‐foot contact in the deceptive and non‐deceptive‐exaggerated conditions, but not the non‐deceptive condition. Because both deceptive and non‐deceptive‐exaggerated kicks contained exaggeration, results suggest exaggerated movements in the kickers’ action at 80 ms before ball‐foot contact explains why a cue‐based mode prevails when anticipating deceptive kicks at this time point.
Intrinsic and extrinsic motivation as predictors of work effort: The moderating role of achievement goals
Authors: Dysvik Anders , Kuvaas Bård
Published: January 2012

This research explored the roles of intrinsic motivation (IM) and extrinsic motivation (EM) and the 2 × 2 model of achievement goals as predictors of increased work effort (WE). A cross‐lagged field study was conducted among 1,441 employees from three large Norwegian service organizations across a 10‐month time span. The results showed that the relationship between IM and increased WE was more positive for employees with high levels of mastery‐approach goals. This observation suggests that having congruent goals may accentuate the positive relationship between IM and WE.
A non-solvable extension of ℚ unramified outside 7
Authors: Dieulefait Luis V.
Published: January 2012

We consider a mod 7 Galois representation attached to a genus 2 Siegel cusp form of level 1 and weight 28 and using some of its Fourier coefficients and eigenvalues computed by N. Skoruppa and the classification of maximal subgroups of PGSp(4,p) we show that its image is as large as possible. This gives a realization of PGSp(4,7) as a Galois group over ℚ and the corresponding number field provides a non-solvable extension of ℚ which ramifies only at 7.
At the heart of computational modelling
Authors: Niederer S. A. , Smith N. P.
Published: January 2012

Abstract  The link between experimental data and biophysically based mathematical models is key to computational simulation meeting its potential to provide physiological insight. However, despite the importance of this link, scrutiny and analysis of the processes by which models are parameterised from data are currently lacking. While this situation is common to many areas of physiological modelling, to provide a concrete context, we use examples drawn from detailed models of cardiac electro‐mechanics. Using this biophysically detailed cohort of models we highlight the specific issues of model parameterization and propose this process can be separated into three stages: observation, fitting and validation. Finally, future research challenges and directions in this area are discussed.
Role of nitric oxide and prostanoids in the regulation of leg blood flow and blood pressure in humans with essential hypertension: effect of high‐intensity aerobic training
Authors: Nyberg Michael , Jensen Lasse G. , Thaning Pia , Hellsten Ylva , Mortensen Stefan P.
Published: January 2012

Key points 

Nitric oxide and prostanoids are substances that dilate the blood vessels. We examined the role of these vasodilators in the regulation of blood flow to contracting muscle and systemic blood pressure before and after a training intervention in subjects with essential hypertension and in healthy controls.

We show that blood flow to the exercising leg is lower in essential hypertension.

Surprisingly, this effect on blood flow is not the result of a reduced capacity of the nitric oxide and prostanoid systems to dilate the blood vessels; however, these systems do appear to play a role in the training induced reduction in blood pressure.

These findings advance our understanding of vascular dysfunction associated with essential hypertension and the mechanisms underlying the blood pressure reducing effect of exercise.
A Three-Dimensional Inverse Finite Element Analysis of the Heel Pad
Authors: Chokhandre Snehal , Halloran Jason P. , van den Bogert Antonie J. , Erdemir Ahmet
Published: January 2012

Quantification of plantar tissue behavior of the heel pad is essential in developing computational models for predictive analysis of preventive treatment options such as footwear for patients with diabetes. Simulation based studies in the past have generally adopted heel pad properties from the literature, in return using heel-specific geometry with material properties of a different heel. In exceptional cases, patient-specific material characterization was performed with simplified two-dimensional models, without further evaluation of a heel-specific response under different loading conditions. The aim of this study was to conduct an inverse finite element analysis of the heel in order to calculate heel-specific material properties in situ. Multidimensional experimental data available from a previous cadaver study by Erdemir et al. (“An Elaborate Data Set Characterizing the Mechanical Response of the Foot,” ASME J. Biomech. Eng., 131(9), pp. 094502) was used for model development, optimization, and evaluation of material properties. A specimen-specific three-dimensional finite element representation was developed. Heel pad material properties were determined using inverse finite element analysis by fitting the model behavior to the experimental data. Compression dominant loading, applied using a spherical indenter, was used for optimization of the material properties. The optimized material properties were evaluated through simulations representative of a combined loading scenario (compression and anterior-posterior shear) with a spherical indenter and also of a compression dominant loading applied using an elevated platform. Optimized heel pad material coefficients were 0.001084 MPa (μ), 9.780 (α) (with an effective Poisson’s ratio (ν) of 0.475), for a first-order nearly incompressible Ogden material model. The model predicted structural response of the heel pad was in good agreement for both the optimization (<1.05% maximum tool force, 0.9% maximum tool displacement) and validation cases (6.5% maximum tool force, 15% maximum tool displacement). The inverse analysis successfully predicted the material properties for the given specimen-specific heel pad using the experimental data for the specimen. The modeling framework and results can be used for accurate predictions of the three-dimensional interaction of the heel pad with its surroundings.
The Biomechanical Effect of Torsion on Humeral Shaft Repair Techniques for Completed Pathological Fractures
Authors: Al-Jahwari Ahmed , Schemitsch Emil H. , Wunder Jay S. , Ferguson Peter C. , Zdero Rad
Published: January 2012

In the presence of a tumor defect, completed humeral shaft fractures continue to be a major surgical challenge since there is no “gold standard” treatment. This is due, in part, to the fact that only one prior biomechanical study exists on the matter, but which only compared 2 repair methods. The current authors measured the humeral torsional performance of 5 fixation constructs for completed pathological fractures. In 40 artificial humeri, a 2-cm hemi-cylindrical cortical defect with a transverse fracture was created in the lateral cortex. Specimens were divided into 5 different constructs and tested in torsion. Construct A was a broad 10-hole 4.5-mm dynamic compression plate (DCP). Construct B was the same as A except that the screw holes and the tumor defect were filled with bone cement and the screws were inserted into soft cement. Construct C was the same as A except that the canal and tumor defect were filled with bone cement and the screws were inserted into dry cement. Construct D was a locked intramedullary nail inserted in the antegrade direction. Construct E was the same as D except that bone cement filled the defect. For torsional stiffness, construct C (4.45 ± 0.20 Nm/deg) was not different than B or E (p > 0.16), but was higher than A and D (p < 0.001). For failure torque, construct C achieved a higher failure torque (69.65 ± 5.35 Nm) than other groups (p < 0.001). For the failure angle, there were no differences between plating constructs A to C (p ≥ 0.11), except for B versus C (p < 0.05), or between nailing groups D versus E (p = 0.97), however, all plating groups had smaller failure angles than both nailing groups (p < 0.05). For failure energy, construct C (17.97 ± 3.59 J) had a higher value than other groups (p < 0.005), except for A (p = 0.057). Torsional failure always occurred in the bone in the classic “spiral” pattern. Construct C provided the highest torsional stability for a completed pathological humeral shaft fracture.
Partial Meniscectomy Changes Fluid Pressurization in Articular Cartilage in Human Knees
Authors: Kazemi M. , Li L. P. , Buschmann M. D. , Savard P.
Published: January 2012

Partial meniscectomy is believed to change the biomechanics of the knee joint through alterations in the contact of articular cartilages and menisci. Although fluid pressure plays an important role in the load support mechanism of the knee, the fluid pressurization in the cartilages and menisci has been ignored in the finite element studies of the mechanics of meniscectomy. In the present study, a 3D fibril-reinforced poromechanical model of the knee joint was used to explore the fluid flow dependent changes in articular cartilage following partial medial and lateral meniscectomies. Six partial longitudinal meniscectomies were considered under relaxation, simple creep, and combined creep loading conditions. In comparison to the intact knee, partial meniscectomy not only caused a substantial increase in the maximum fluid pressure but also shifted the location of this pressure in the femoral cartilage. Furthermore, these changes were positively correlated to the size of meniscal resection. While in the intact joint, the location of the maximum fluid pressure was dependent on the loading conditions, in the meniscectomized joint the location was predominantly determined by the site of meniscal resection. The partial meniscectomy also reduced the rate of the pressure dissipation, resulting in even larger difference between creep and relaxation times as compared to the case of the intact knee. The knee joint became stiffer after meniscectomy because of higher fluid pressure at knee compression followed by slower pressure dissipation. The present study indicated the role of fluid pressurization in the altered mechanics of meniscectomized knees.
Magnetic anisotropy of GaAs/Fe/Au core-shell nanowires grown by MBE
Authors: Tivakornsasithorn K. , Pimpinella R. E. , Nguyen V. , Liu X. , Dobrowolska M. , Furdyna J. K.
Published: January 2012

GaAs/Fe/Au core-shell nanowires were grown on GaAs(111)B substrates by molecular beam epitaxy. Scanning electron microscopy images show that the Fe shell has successfully coated the sidewalls of GaAs nanowires. Magnetic anisotropy of GaAs/Fe core-shell nanowires was studied by ferromagnetic resonance and by superconducting quantum interference device magnetometer. The authors’ results show that the magnetic anisotropy of this novel core-shell nanowire system cannot be simply described by any known theory, as revealed by attempts to use micromagnetic simulation using the Object Oriented MicroMagnetic Framework. The observed features thus suggest the existence of a domain structure that is specific to this new system
High rate roll to roll atomic layer deposition, and its application to moisture barriers on polymer films
Authors: Dickey Eric , Barrow William A.
Published: January 2012

Atomic layer deposition has been shown to provide high quality single layer moisture barrier films on polymer substrates, but conventional pulse-based processes are too slow to be commercially feasible. One way to overcome this speed limitation is to avoid the need to pulse and purge precursors by moving the substrate between zones containing the precursors, passing through intermediate purge zones. Recently, several groups have reported various approaches to accomplishing this, including the approach discussed here in which the flexible web is passed between precursor zones in a serpentine pattern. Al2O3 and TiO2 barrier films 12 to 20 nm thick with water vapor transmission rates in the range of 10−4 g/m2/day have been demonstrated for web speeds in excess of 1 m/s on 100 mm wide polyethylene terephthalate web. Scale-up of this process to 300 mm wide web in a system capable of depositing 10–20 nm of film in a single pass is currently under way. This scale-up effort and the potential for very high volume, low cost moisture barrier production utilizing this technique are discussed.
Extracellular Ca
2+
Acts as a Mediator of Communication from Neurons to Glia
Authors: Torres Arnulfo , Wang Fushun , Xu Qiwu , Fujita Takumi , Dobrowolski Radoslaw , Willecke Klaus , Takano Takahiro , Nedergaard Maiken
Published: January 2012

Local decreases in extracellular calcium trigger signaling events in astrocytes that activate inhibitory neurotransmission.
Natural Killer Controls
Authors: Mueller Kristen L.
Published: January 2012

The activating receptor NKp46 is important for keeping the responses of natural killer cells in check.
First Sulfhydration, Then Nitrosylation
Authors: Gough Nancy R.
Published: January 2012

Sulfydration of a subunit of NF-κB promotes its interaction with a coactivator and stimulates production of genes encoding antiapoptotic proteins.
Significant Reduction of Brain Cysts Caused by Toxoplasma gondii after Treatment with Spiramycin Coadministered with Metronidazole in a Mouse Model of Chronic Toxoplasmosis
Authors: Chew Wai Kit , Segarra Ignacio , Ambu Stephen , Mak Joon Wah
Published: January 2012

Toxoplasma gondii
is a parasite that generates latent cysts in the brain; reactivation of these cysts may lead to fatal toxoplasmic encephalitis, for which treatment remains unsuccessful. We assessed spiramycin pharmacokinetics coadministered with metronidazole, the eradication of brain cysts and the
in vitro
reactivation. Male BALB/c mice were fed 1,000 tachyzoites orally to develop chronic toxoplasmosis. Four weeks later, infected mice underwent different treatments: (i) infected untreated mice (
n
= 9), which received vehicle only; (ii) a spiramycin-only group (
n
= 9), 400 mg/kg daily for 7 days; (iii) a metronidazole-only group (
n
= 9), 500 mg/kg daily for 7 days; and (iv) a combination group (
n
= 9), which received both spiramycin (400 mg/kg) and metronidazole (500 mg/kg) daily for 7 days. An uninfected control group (
n
= 10) was administered vehicle only. After treatment, the brain cysts were counted, brain homogenates were cultured in confluent Vero cells, and cysts and tachyzoites were counted after 1 week. Separately, pharmacokinetic profiles (plasma and brain) were assessed after a single dose of spiramycin (400 mg/kg), metronidazole (500 mg/kg), or both. Metronidazole treatment increased the brain spiramycin area under the concentration-time curve from 0 h to ∞ (AUC
0–∞
) by 67% without affecting its plasma disposition. Metronidazole plasma and brain AUC
0–∞
values were reduced 9 and 62%, respectively, after spiramycin coadministration. Enhanced spiramycin brain exposure after coadministration reduced brain cysts 15-fold (79 ± 23 for the combination treatment versus 1,198 ± 153 for the untreated control group [
P
< 0.05]) and 10-fold versus the spiramycin-only group (768 ± 125). Metronidazole alone showed no effect (1,028 ± 149). Tachyzoites were absent in the brain. Spiramycin reduced
in vitro
reactivation. Metronidazole increased spiramycin brain penetration, causing a significant reduction of
T. gondii
brain cysts, with potential clinical translatability for chronic toxoplasmosis treatment.
Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice.
Authors: Brooks A R , Nagy B P , Taylor S , Simonet W S , Taylor J M , Levy-Wilson B
Published: January 2012

To identify DNA sequence elements from the human apolipoprotein B (apoB) gene required for high-level, correct tissue-specific expression in transgenic mice, we made several constructs that included one or more of the key regulatory elements that were previously characterized with cultured liver-derived and intestine-derived cell lines. Our data show that the apoB promoter alone (-898 to +121) is not sufficient to direct transcription in transgenic mice. An enhancer located in the second intron is absolutely required to specify transcription by the homologous apoB promoter in the livers of transgenic mice; this enhancer does not direct transcription in the small intestines. Thus, the elements controlling transcriptional activation of the apoB gene in the liver and the intestine in vivo are distinct and separable. Analysis of the DNase I hypersensitivity of the integrated human transgenes in various lines of expressing and nonexpressing mice suggests that the formation of DH4, a strong hypersensitive site in intron 2, may be a prerequisite for hepatic expression of the apoB gene. Nuclear matrix association regions (MARs) of the apoB gene may play a role in transgene expression. Constructs including MAR sequences displayed higher levels of expression than those lacking them. However, these MARs did not completely insulate the associated transgenes from position effects.
A gene involved in control of human cellular senescence on human chromosome 1q.
Authors: Hensler P J , Annab L A , Barrett J C , Pereira-Smith O M
Published: January 2012

Normal cells in culture exhibit limited division potential and have been used as a model for cellular senescence. In contrast, tumor-derived or carcinogen- or virus-transformed cells are capable of indefinite division. Fusion of normal human diploid fibroblasts with immortal human cells yielded hybrids having limited life spans, indicating that cellular senescence was dominant. Fusions of various immortal human cell lines with each other led to the identification of four complementation groups for indefinite division. The purpose of this study was to determine whether human chromosome 1 could complement the recessive immortal defect of human cell lines assigned to one of the four complementation groups. Using microcell fusion, we introduced a single normal human chromosome 1 into immortal human cell lines representing the complementation groups and determined that it caused loss of proliferative potential of an osteosarcoma-derived cell line (TE85), a cytomegalovirus-transformed lung fibroblast cell line (CMV-Mj-HEL-1), and a Ki-ras(+)-transformed derivative of TE85 (143B TK-), all of which were assigned to complementation group C. This chromosome 1 caused no change in proliferative potential of cell lines representing the other complementation groups. A derivative of human chromosome 1 that had lost most of the q arm by spontaneous deletion was unable to induce senescence in any of the immortal cell lines. This finding indicates that the q arm of human chromosome 1 carries a gene or set of genes which is altered in the cell lines assigned to complementation group C and is involved in the control of cellular senescence.
Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.
Authors: Tate B F , Allenby G , Janocha R , Kazmer S , Speck J , Sturzenbecker L J , Abarzúa P , Levin A A , Grippo J F
Published: January 2012

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.
Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.
Authors: van den Berghe N , Ouwens D M , Maassen J A , van Mackelenbergh M G , Sips H C , Krans H M
Published: January 2012

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.
Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.
Authors: Philip R , Brunette E , Kilinski L , Murugesh D , McNally M A , Ucar K , Rosenblatt J , Okarma T B , Lebkowski J S
Published: January 2012

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.
Non-Mendelian inheritance of macronuclear mutations is gene specific in Paramecium tetraurelia.
Authors: Scott J M , Mikami K , Leeck C L , Forney J D
Published: January 2012

Paramecium tetraurelia contains two types of nuclei, a diploid germinal micronucleus and a large transcriptionally active macronucleus. The macronuclear genome is formed from the micronuclear DNA during sexual reproduction. Previous studies have shown that the processing of the A-type variable surface protein gene during formation of a new macronucleus is dependent on the presence of the A gene in the old macronucleus. It is not clear if this is a general feature that controls the formation of the Paramecium macronuclear genome or a unique feature of the A locus. Using micronuclear transplantation, we have constructed a strain that has a wild-type micronucleus but has macronuclear deletions of the A- and B-type surface protein genes. Neither the A nor the B gene is incorporated into the new macronucleus after sexual reproduction. Macronuclear transformation of this strain with the B gene rescues the B-gene deletion after formation of the next macronucleus but has not effect on the A deletion. Similarly, transformation with the A gene shows gene-specific rescue for A but not B. The effect of the old macronucleus on the processing of the new macronucleus results in a pattern of non-Mendelian inheritance of both macronuclear deletions. Progeny from the wild-type exconjugant are all wild type, and progeny from the A- B- exconjugant are mutant. The features of this A- B- non-Mendelian mutant demonstrate that the regulation of macronuclear DNA processing is gene specific, and our results open the possibility that this type of regulation affects many regions of the Paramecium genome.
Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.
Authors: Songyang Z , Shoelson S E , McGlade J , Olivier P , Pawson T , Bustelo X R , Barbacid M , Sabe H , Hanafusa H , Yi T
Published: January 2012

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
Histone H1 expressed in Saccharomyces cerevisiae binds to chromatin and affects survival, growth, transcription, and plasmid stability but does not change nucleosomal spacing.
Authors: Linder C , Thoma F
Published: January 2012

Histone H1 is proposed to serve a structural role in nucleosomes and chromatin fibers, to affect the spacing of nucleosomes, and to act as a general repressor of transcription. To test these hypotheses, a gene coding for a sea urchin histone H1 was expressed from the inducible GAL1 promoter in Saccharomyces cerevisiae by use of a YEp vector for high expression levels (strain YCL7) and a centromere vector for low expression levels (strain YCL1). The H1 protein was identified by its inducibility in galactose, its apparent molecular weight, and its solubility in 5% perchloric acid. When YCL7 was shifted from glucose to galactose for more than 40 h to achieve maximal levels of H1, H1 could be copurified in approximately stoichiometric amounts with core histones of Nonidet P-40-washed nuclei and with soluble chromatin fractionated on sucrose gradients. While S. cerevisiae tolerated the expression of low levels of H1 in YCL1 without an obvious phenotype, the expression of high levels of H1 correlated with greatly reduced survival, inhibition of growth, and increased plasmid loss but no obvious change in the nucleosomal repeat length. After an initial induction, RNA levels for GAL1 and H1 were drastically reduced, suggesting that H1 acts by the repression of galactose-induced genes. Similar effects, but to a lower extent, were observed when the C-terminal tail of H1 was expressed.
Down-regulation of nuclear aryl hydrocarbon receptor DNA-binding and transactivation functions: requirement for a labile or inducible factor.
Authors: Reick M , Robertson R W , Pasco D S , Fagan J B
Published: January 2012

Aryl hydrocarbons (AHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene activate the sequence-specific DNA-binding activity of the AH receptor. In the rat hepatocyte-derived cell line LCS7, DNA-binding activity peaked after 30 min and was then down-regulated, reaching negligible levels by 2 h. Down-regulation could be blocked, and DNA-binding activity maintained at maximum for many hours by inhibiting protein or RNA synthesis, implying that down-regulation is a mediated process requiring a labile or inducible protein. CYP1A1 transcription and in vivo DNA-protein interactions at xenobiotic response elements were down-regulated in parallel with DNA-binding activity in nuclear extracts, and these changes could also be blocked by inhibitors of protein synthesis. The correlation between AH receptor DNA-binding activity, intensity of in vivo footprints at xenobiotic response elements, and CYP1A1 transcription rate implies that down-regulation of AH receptor DNA-binding activity is important in regulating CYP1A1 transcription and that receptor is required continuously to maintain transcription. This correlation extends to the murine hepatoma cell line Hepa-1c1c7, in which slower kinetics of activation and down-regulation of CYP1A1 transcription paralleled slower activation and down-regulation of AH receptor DNA-binding activity. The difference in kinetics between cell lines also implies that AH receptor DNA-binding activity is modulated by a mechanism that may be influenced by cell-specific regulatory pathways. The above observations in conjunction with mixing experiments and comparisons of cytoplasmic and nuclear extracts indicate that down-regulation of AH receptor DNA-binding activity is probably due either to degradation or to conversion of the receptor to form that is inactive in both DNA binding and transactivation.
The delta-crystallin enhancer-binding protein delta EF1 is a repressor of E2-box-mediated gene activation.
Authors: Sekido R , Murai K , Funahashi J , Kamachi Y , Fujisawa-Sehara A , Nabeshima Y , Kondoh H
Published: January 2012

The repressor delta EF1 was discovered by its action on the DC5 fragment of the lens-specific delta 1-crystallin enhancer. C-proximal zinc fingers of delta EF1 were found responsible for binding to the DC5 fragment and had specificity to CACCT as revealed by selection of high-affinity binding sequences from a random oligonucleotide pool. CACCT is present not only in DC5 but also in the E2 box (CACCTG) elements which are the binding sites of various basic helix-loop-helix activators and also the target of an unidentified repressor, raising the possibility that delta EF1 accounts for the E2 box repressor activity. delta EF1 competed with E47 for binding to an E2 box sequence in vitro. In lymphoid cells, endogenous delta EF1 activity as a repressor was detectable, and exogenous delta EF1 repressed immunoglobulin kappa enhancer by binding to the kappa E2 site. Moreover, delta EF1 repressed MyoD-dependent activation of the muscle creatine kinase enhancer and MyoD-induced myogenesis of 10T1/2 cells. Thus, delta EF1 counteracts basic helix-loop-helix activators through binding site competition and fulfills the conditions of the E2 box repressor. In embryonic tissues, the most prominent site of delta EF1 expression is the myotome. Myotomal expression as well as the above results argues for a significant contribution of delta EF1 in regulation of embryonic myogenesis through the modulation of the actions of MyoD family proteins.
Cooperativity between two NF-kappa B complexes, mediated by high-mobility-group protein I(Y), is essential for cytokine-induced expression of the E-selectin promoter.
Authors: Lewis H , Kaszubska W , DeLamarter J F , Whelan J
Published: January 2012

Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-140/-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-kappa B consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-kappa B-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-kappa B to both NF-kappa B sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-selectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-kappa B complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-kappa B complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-kappa B complexes are involved in specific gene activation.
Ty1 in vitro integration: effects of mutations in cis and in trans.
Authors: Braiterman L T , Boeke J D
Published: January 2012

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.
Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.
Authors: Packham G , Cleveland J L
Published: January 2012

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.
Effects of c-myc expression on cell cycle progression.
Authors: Hanson K D , Shichiri M , Follansbee M R , Sedivy J M
Published: January 2012

We used targeted homologous recombination to disrupt one c-myc gene copy in a diploid fibroblast cell line and found that a twofold reduction in Myc expression resulted in lower exponential growth rates and a lengthening of the G0-to-S-phase transition (M. Shichiri, K. D. Hanson and J. M. Sedivy, Cell Growth Differ. 4:93-104, 1993). Myc is a transcription factor, and the number of target genes whose regulation could result in differential growth rates may be very large. We have approached this problem by examining effects of reduced c-myc expression in three broad areas: (i) secretion of growth factors, (ii) expression of growth factor receptors, and (iii) intracellular signal transduction between Myc and components of the intrinsic cell cycle clock. We have found no evidence that differential medium conditioning can account for the growth phenotypes. Likewise, the expression of receptors for platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor I was the same in diploid and heterozygous cells (platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, and insulin-like growth factor are the sole growth factors required by these cells for growth in serum-free medium). In contrast, expression of cyclin E, cyclin A, and Rb phosphorylation were delayed when quiescent c-myc heterozygous cells were stimulated to enter the cell cycle. Expression of cyclin D1, cyclin D3, and Cdk2 was not affected. The timing of cyclin E induction was the earliest observable effect of reduced Myc expression. Our data indicate that Myc contributes to regulation of proliferation by a cell-autonomous mechanism that involves the modulation of cyclin E expression and, consequently, progression through the restriction point of the cell cycle.
Unusual chromatin in human telomeres.
Authors: Tommerup H , Dousmanis A , de Lange T
Published: January 2012

We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.
Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.
Authors: Phillips J W , Morgan W F
Published: January 2012

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.
The PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable.
Authors: Janz L , Clayton C
Published: January 2012

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.