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1

Genetic analysis of basophil function in vivo  

PubMed Central

Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive IL-4 reporter alleles, we demonstrate that basophil IL-4 production occurs by a CD4+ T cell-dependent process restricted to affected peripheral tissues. We genetically marked and specifically deleted basophils and demonstrate that basophils do not mediate TH2 priming in vivo. Two-photon imaging confirmed that basophils do not interact with antigen-specific T cells in lymph nodes, but can engage in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4 and IL-13 in either CD4+ T cells or basophils minimally impacted worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity.

Sullivan, Brandon M.; Liang, Hong-Erh; Bando, Jennifer K.; Wu, Davina; Cheng, Laurence E.; McKerrow, James K.; Allen, Christopher D. C.; Locksley, Richard M.

2012-01-01

2

Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression  

Microsoft Academic Search

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease

Weisheng Zhang; Jian Q. Feng; Stephen E. Harris; Pamela R. Contag; David K. Stevenson; Christopher H. Contag

2001-01-01

3

In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites  

PubMed Central

Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs.

Hertz, Marla I.; Thompson, Sunnie R.

2011-01-01

4

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila  

PubMed Central

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.

Reis, Gerald F.; Yang, Ge; Szpankowski, Lukasz; Weaver, Carole; Shah, Sameer B.; Robinson, John T.; Hays, Thomas S.; Danuser, Gaudenz; Goldstein, Lawrence S. B.

2012-01-01

5

In Vivo Analysis of Lrig Genes Reveals Redundant and Independent Functions in the Inner Ear  

PubMed Central

Lrig proteins are conserved transmembrane proteins that modulate a variety of signaling pathways from worm to humans. In mammals, there are three family members – Lrig1, Lrig2, and Lrig3 – that are defined by closely related extracellular domains with a similar arrangement of leucine rich repeats and immunoglobulin domains. However, the intracellular domains show little homology. Lrig1 inhibits EGF signaling through internalization and degradation of ErbB receptors. Although Lrig3 can also bind ErbB receptors in vitro, it is unclear whether Lrig2 and Lrig3 exhibit similar functions to Lrig1. To gain insights into Lrig gene functions in vivo, we compared the expression and function of the Lrigs in the inner ear, which offers a sensitive system for detecting effects on morphogenesis and function. We find that all three family members are expressed in the inner ear throughout development, with Lrig1 and Lrig3 restricted to subsets of cells and Lrig2 expressed more broadly. Lrig1 and Lrig3 overlap prominently in the developing vestibular apparatus and simultaneous removal of both genes disrupts inner ear morphogenesis. This suggests that these two family members act redundantly in the otic epithelium. In contrast, although Lrig1 and Lrig2 are frequently co-expressed, Lrig1?/?;Lrig2?/? double mutant ears show no enhanced structural abnormalities. At later stages, Lrig1 expression is sustained in non-sensory tissues, whereas Lrig2 levels are enhanced in neurons and sensory epithelia. Consistent with these distinct expression patterns, Lrig1 and Lrig2 mutant mice exhibit different forms of impaired auditory responsiveness. Notably, Lrig1?/?;Lrig2?/? double mutant mice display vestibular deficits and suffer from a more severe auditory defect that is accompanied by a cochlear innervation phenotype not present in single mutants. Thus, Lrig genes appear to act both redundantly and independently, with Lrig2 emerging as the most functionally distinct family member.

del Rio, Tony; Nishitani, Allison M.; Yu, Wei-Ming; Goodrich, Lisa V.

2013-01-01

6

In vivo analysis of the functional domains of the Drosophila splicing regulator RBP1  

PubMed Central

The Drosophila splicing factor RBP1 participates together with TRA and TRA-2 in the regulation of alternative splicing of doublesex (dsx) pre-mRNA. It does so by recognizing RBP1 RNA target sequences in the dsx pre-mRNA. RBP1 belongs to the Ser–Arg-rich (SR) protein family of splicing factors, which have in common a N-terminal RNA recognition motif-type RNA binding domain, a Gly-rich region, and a C-terminal SR domain. Using a tissue culture transfection assay, we demonstrate that the Gly residues within the Gly-rich domain, the ribonucleoprotein motifs within the RNA recognition motif RNA binding domain, and the SR domain are required for regulation of dsx splicing by RBP1 in vivo. Furthermore, using a two-hybrid system, we show protein–protein interactions between RBP1 and itself and between RBP1 and TRA-2. The SR domain and the Gly residues within the Gly-rich domain of RBP1 were found to be involved in these protein–protein interactions. Our results suggest that RBP1 and TRA-2 function in regulation of dsx splicing by forming a complex.

Heinrichs, Volker; Baker, Bruce S.

1997-01-01

7

In vivo analysis of p53 tumor suppressor function using genetically engineered mouse models  

PubMed Central

p53 is a crucial tumor suppressor, as evidenced by the high propensity for p53 mutation during human cancer development. Already more than a decade ago, p53 knockout mice confirmed that p53 is critical for preventing tumorigenesis. More recently, a host of p53 knock-in mouse strains has been generated, with the aim of either more precisely modeling p53 mutations in human cancer or better understanding p53's regulation and downstream activities. In the first category, several mouse strains expressing mutant p53 proteins corresponding to human-tumor-derived mutants have demonstrated that mutant p53 is not equivalent to loss of p53 but additionally exhibits gain-of-function properties, promoting invasive and metastatic phenotypes. The second class of p53 knock-in mouse models expressing engineered p53 mutants has also provided new insight into p53 function. For example, mice expressing p53 mutants lacking specific posttranslational modification sites have revealed that these modifications serve to modulate p53 responses in vivo in a cell-type- and stress-specific manner rather than being absolutely required for p53 stabilization and activation as suggested by in vitro experiments. Additionally, studies of p53 mouse models have established that both p53-driven cell-cycle arrest and apoptosis responses contribute to tumor suppression and that activation of p53 by oncogenic stress imposes an important barrier to tumorigenesis. Finally, the use of mouse strains expressing temporally regulatable p53 has demonstrated that p53 loss is not only required for tumor development but also required for tumor maintenance, suggesting that p53 restoration in human cancer patients may be a promising therapeutic strategy. These sophisticated p53 mouse models have taught us important lessons, and new mouse models will certainly continue to reveal interesting and perhaps surprising aspects of p53's complex biology.

Broz, Daniela Kenzelmann; Attardi, Laura D.

2010-01-01

8

In vivo analysis of the functions of gamma-tubulin-complex proteins.  

PubMed

To enhance our understanding of the function(s) of gamma-tubulin-complex proteins (GCPs), we identified and analyzed the functions of the Aspergillus nidulans homologs of GCP2-GCP6 (here designated GCPB-GCBF). The gamma-tubulin small complex (gamma-TuSC) components, gamma-tubulin, GCPB and GCPC, are essential for viability and mitotic spindle formation, whereas GCPD-GCPF are not essential for viability, spindle formation or sexual reproduction. GCPD-GCPF function in reducing the frequency of chromosome mis-segregation and in the assembly of large gamma-tubulin complexes. Deletion of any of the gamma-TuSC components eliminates the localization of all GCPs to the spindle pole body (SPB), whereas deletion of GCPD-GCPF does not affect localization of gamma-TuSC components. Thus, GCPD-GCPF do not tether the gamma-TuSC to the SPB, but, rather, the gamma-TuSC tethers them to the SPB. GCPD-GCPF exhibit a hierarchy of localization to the SPB. Deletion of GCPF eliminates GCPD-GCPE localization to the SPB, and deletion of GCPD eliminates GCPE (but not GCPF) localization. All GCPs localize normally in a GCPE deletion. We propose a model for the structure of the gamma-tubulin complex and its attachment to polar microtubule organizing centers. PMID:19861490

Xiong, Yi; Oakley, Berl R

2009-10-27

9

An in vivo analysis of the vestigial gene in Drosophila melanogaster defines the domains required for Vg function.  

PubMed Central

Considerable evidence indicates an obligate partnership of the Drosophila melanogaster Vestigial (VG) and Scalloped (SD) proteins within the context of wing development. These two proteins interact physically and a 56-amino-acid motif within VG is necessary and sufficient for this binding. While the importance of this SD-binding domain has been clearly demonstrated both in vitro and in vivo, the remaining portions of VG have not been examined for in vivo function. Herein, additional regions within VG were tested for possible in vivo functions. The results identify two additional domains that must be present for optimal VG function as measured by the loss of ability to rescue vg mutants, to induce ectopic sd expression, and to perform other normal VG functions when they are deleted. An in vivo study such as this one is fundamentally important because it identifies domains of VG that are necessary in the cellular context in which wing development actually occurs. The results also indicate that an additional large portion of VG, outside of these two domains and the SD-binding domain, is dispensable in the execution of these normal VG functions.

MacKay, Julie O; Soanes, Kelly H; Srivastava, Ajay; Simmonds, Andrew; Brook, William J; Bell, John B

2003-01-01

10

Reversal of coenzyme specificity of 2,3-butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis.  

PubMed

Saccharomyces cerevisiae NAD(H)-dependent 2,3-butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)-dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu(221), Ile(222), and Ala(223)) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site-directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration. PMID:19507198

Ehsani, Maryam; Fernández, Maria R; Biosca, Josep A; Dequin, Sylvie

2009-10-01

11

First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome.  

PubMed

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease. PMID:23954224

Ben Mahmoud, Afif; Siala, Olfa; Mansour, Riadh Ben; Driss, Fatma; Baklouti-Gargouri, Siwar; Mkaouar-Rebai, Emna; Belguith, Neila; Fakhfakh, Faiza

2013-08-14

12

Assessment of functional knee bracing: an in vivo three-dimensional kinematic analysis of the anterior cruciate deficient knee  

Microsoft Academic Search

Objective. To describe three-dimensional tibial and femoral movements in vivo and examine the effect of a brace on knee kinematics during moderate to intense activity.Design. Skeletal kinematics of anterior cruciate ligament deficient knees was measured with and without braces during moderate to intense activity.Background. Invasive markers implanted into the tibia and femur are the most accurate means to directly measure

Dan K Ramsey; Mario Lamontagne; Per F Wretenberg; Anders Valentin; Björn Engström; Gunnar Németh

2001-01-01

13

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins  

Microsoft Academic Search

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH\\/MIH\\/GIH family. Until now the structure–function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the

Romina Mettulio; Piero Giulio Giulianini; Enrico Antonio Ferrero; Simonetta Lorenzon; Paolo Edomi

2004-01-01

14

Proteomic analysis of the function of spot in Helicobacter pylori anti-oxidative stress in vitro and colonization in vivo.  

PubMed

As a microaerobe, Helicobacter pylori employs the global regulator SpoT for defending against oxidative stress in vitro. However, the mechanisms how SpoT affects bacterial gene expression is still unknown. Moreover, the function of SpoT in H. pylori colonization in the host is remaining undetermined. To explore the functions of the SpoT in H. pylori pathogenesis, we constructed H. pylori 26695 spoT-deficient mutant (?spoT). While grown in ambient atmosphere, protein expression profile of the ?spoT was analyzed with 2D gel electrophoresis and real-time PCR. Compared to the wild type, the spoT-deficient strain downregulated its transcription of the oxidative-induced genes, as well as the genes responsible for protein degradation and that related to energy metabolism. Meanwhile, the colonization ability of ?spoT strains in Mongolian gerbil was tested, the results demonstrated a decayed colonization in the mouse stomach with ?spoT than the wild type. As a matter of facts, the AGS cells infected with the ?spoT strains excreted increased level of the gastric inflammation cytokines IL-8, and the ?spoT strains showed poor survival ability when treated with reactive oxygen stress (sodium nitroprusside). The elevated capacity of stimulating cytokines and fragility to reactive oxygen stress may be contribute to decreased colonization of the spoT-deficient mutant in the mouse stomach. Conclusively, we speculate that spoT is a key regulator of the genes for H. pylori spreading in the air and colonization in host stomach. PMID:22678710

Sun, Yundong; Li, Xinpeng; Li, Wen; Zhao, Min; Wang, Lixiang; Liu, Shili; Zeng, Jiping; Liu, Zhifang; Jia, Jihui

2012-11-01

15

Cyclin D1 Determines Mitochondrial Function In Vivo  

PubMed Central

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

Sakamaki, Toshiyuki; Casimiro, Mathew C.; Ju, Xiaoming; Quong, Andrew A.; Katiyar, Sanjay; Liu, Manran; Jiao, Xuanmao; Li, Anping; Zhang, Xueping; Lu, Yinan; Wang, Chenguang; Byers, Stephen; Nicholson, Robert; Link, Todd; Shemluck, Melvin; Yang, Jianguo; Fricke, Stanley T.; Novikoff, Phyllis M.; Papanikolaou, Alexandros; Arnold, Andrew; Albanese, Christopher; Pestell, Richard

2006-01-01

16

EPR Spectroscopy of Function In Vivo  

Microsoft Academic Search

EPR can be used to study free radicals in vivo, environmental and biophysical parameters in cells and tissues, and to report metabolism, physiology, and biochemistry. The authors have attempted to judge which of these types of measurements will be productive for studies in animals and in humans. It is envisioned that a large number of in vivo applications of EPR

Harold M. Swartz; Nadeem Khan

17

Estimating the input function non-invasively for FDG-PET quantification with multiple linear regression analysis: simulation and verification with in vivo data  

Microsoft Academic Search

A novel statistical method, namely Regression-Estimated Input Function (REIF), is proposed in this study for the purpose of non-invasive estimation of the input function for fluorine-18 2-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) quantitative analysis. We collected 44 patients who had undergone a blood sampling procedure during their FDG-PET scans. First, we generated tissue time-activity curves of the grey matter and the

Yu-Hua Fang; Tsair Kao; Ren-Shyan Liu; Liang-Chih Wu

2004-01-01

18

An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA.  

PubMed

The structure and accessibility of the S. cerevisiae U3A snoRNA was studied in semi-purified U3A snoRNPs using both chemical and enzymatic probes and in vivo using DMS as the probe. The results obtained show that S. cerevisiae U3A snoRNA is composed of a short 5' domain with two stem-loop structures containing the phylogenetically conserved boxes A' and A and a large cruciform 3' domain containing boxes B, C, C' and D. A precise identification of RNA-protein contacts is provided. Protection by proteins in the snoRNP and in vivo are nearly identical and were exclusively found in the 3' domain. There are two distinct protein anchoring sites: (i), box C' and its surrounding region, this site probably includes box D, (ii) the boxes B and C pair and the bases of stem-loop 2 and 4. Box C' is wrapped by the proteins. RNA-protein interactions are more loose at the level of boxes C and D and a box C and D interaction is preserved in the snoRNP. In accord with this location of the protein binding sites, an in vivo mutational analysis showed that box C' is important for U3A snoRNA accumulation, whereas mutations in the 5' domain have little effect on RNA stability. Our in vivo probing experiments strongly suggest that, in exponentially growing cells, most of the U3A snoRNA molecules are involved in the 10-bp interaction with the 5'-ETS region and in two of the interactions recently proposed with 18S rRNA sequences. Our experimental study leads to a slightly revised version of the model of interaction proposed by J. Hughes. Single-stranded segments linking the heterologous helices are highly sensitive to DMS in vivo and their functional importance was tested by a mutational analysis. PMID:9356246

Méreau, A; Fournier, R; Grégoire, A; Mougin, A; Fabrizio, P; Lührmann, R; Branlant, C

1997-10-31

19

Construction of an in vivo System for Functional Analysis of the Genes Involved in Sex Pheromone Production in the Silkmoth, Bombyx mori  

PubMed Central

Moths produce species-specific sex pheromones to attract conspecific mates. The biochemical processes that comprise sex pheromone biosynthesis are precisely regulated and a number of gene products are involved in this biosynthesis and regulation. In recent years, at least 300 EST clones have been isolated from Bombyx mori pheromone gland (PG) specific cDNA libraries with some of those clones [i.e., B. mori PG-specific desaturase 1 (Bmpgdesat1), PG-specific fatty acyl reductase, PG-specific acyl-CoA-binding protein, B. mori fatty acid transport protein, B. mori lipid storage droplet protein-1] characterized and demonstrated to play a role in sex pheromone production. However, most of the EST clones have yet to be fully characterized and identified. To develop an efficient system for analyzing sex pheromone production-related genes, we investigated the feasibility of a novel gene analysis system using the upstream region of Bmpgdesat1 that should contain a PG-specific gene promoter in conjunction with piggyBac vector-mediated germ line transformation. As a result, we have been able to obtain expression of our reporter gene (enhanced green fluorescent protein) in the PG but not in other tissues of transgenic B. mori. Current results indicate that we have successfully constructed a novel in vivo gene analysis system for sex pheromone production in B. mori.

Moto, Ken-Ichi; Matsumoto, Shogo

2012-01-01

20

Spectroscopy analysis of tissues in vivo  

NASA Astrophysics Data System (ADS)

The spectral analysis of biological tissues in vivo is widely used in various fields particularly in medical diagnostics and therapy control. Great possibilities of spectral tissue analysis exist to be realized in the future. Among them are the complete non-invasive clinical blood analysis with evaluation of, for example, sugar concentration in blood; the evaluation of chemical state and localization on subcell level of various drugs binded with biological structures. These facts were shown to affect drastically the drug therapeutic activity. The main advantage of spectral analysis of tissues in vivo is its noninvasivity. This allows one to get information about tissue condition without affecting the dynamic of various biological processes. Another advantage of optical tissue analysis is the possibility to process data in real time and to control parameters of therapy process according to information acquired. For example the in situ analysis of photosensitizer concentration and its chemical state during photodynamic therapy makes it possible to correct the laser irradiation intensity (the photobleaching of photosensitizer requires the decrease in laser intensity).

Loschenov, Victor B.; Poleshkin, P. V.; Stratonnikov, A. A.; Torshina, Nadezgda L.

1995-01-01

21

Sequence analysis of the ?2B-ADRENERGIC receptor (ADRA2B) gene and in-vivo functional effects of novel variants  

Microsoft Academic Search

Background: ADRA2B is important in vasoconstriction and blood pressure regulation. The only common variant identified (del301–303) decreased receptor desensitization in vitro but did not alter vascular sensitivity in vivo. We therefore characterized the genetic variation in ADRA2B and related this to in vivo phenotype.Methods: We examined 5812 bp of contiguous sequence of ADRA2B (promoter, exon, and 3' flanking region) using

M. Muszkat; G. G. Sofowora; D. Kurnik; J. Solus; L. Jiang; S. M. Williams; E. Dawson; A. J. Wood; C. M. Stein

2005-01-01

22

Noninvasive laser-induced photoacoustic tomography for structural and functional in vivo imaging of the brain  

Microsoft Academic Search

Imaging techniques based on optical contrast analysis can be used to visualize dynamic and functional properties of the nervous system via optical signals resulting from changes in blood volume, oxygen consumption and cellular swelling associated with brain physiology and pathology. Here we report in vivo noninvasive transdermal and transcranial imaging of the structure and function of rat brains by means

Xueding Wang; Yongjiang Pang; Geng Ku; Xueyi Xie; George Stoica; Lihong V Wang

2003-01-01

23

Mitochondrial Metabolic Function Assessed In Vivo and In Vitro  

PubMed Central

Purpose of review Mitochondrial content and function vary across species, tissue types, and lifespan. Alterations in skeletal muscle mitochondrial function have been reported to occur in in aging and in many other pathological conditions. This review focuses on the state of the art in vivo and in vitro methodologies for assessment of muscle mitochondrial function. Recent findings Classic studies of isolated mitochondria have measured function from maximal respiratory capacity. These fundamental methods have recently been substantially improved and novel approaches to asses mitochondrial functions in vitro have been emerged. Non-invasive methods based on magnetic resonance spectroscopy (MRS) and near-infrared spectroscopy (NIRS) permit in vivo assessment of mitochondrial function and are rapidly becoming more accessible to many investigators. Moreover, it is now possible to gather information on regulation of mitochondrial content by measuring the in vivo synthesis rate of individual mitochondrial proteins. Summary High-resolution respirometry has emerged as a powerful tool for in vitro measurements of mitochondrial function in isolated mitochondria and permeabilized fibers. Direct measurements of ATP production are possible by bioluminescence. Mechanistic data provided by these methods is further complimented by in vivo assessment using MRS and NIRS and the translational rate of gene transcripts.

Lanza, Ian R.; Nair, K. Sreekumaran

2011-01-01

24

In Vivo Evidence for and Consequences of Functional Selectivity  

Microsoft Academic Search

Functional selectivity refers to the ability of some ligands to stimulate a subset of the possible consequences of activation\\u000a of a receptor. This chapter addresses two related issues that are critical for consideration of the therapeutic utility of\\u000a functional selectivity: the evidence that functional selectivity is a pharmacologically relevant phenomenon that can be observed\\u000a in vivo, and characterization of the

Kim A. Neve; Marc G. Caron; Jean-Martin Beaulieu

25

Resurrection of DNA function in vivo from an extinct genome.  

PubMed

There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity. PMID:18493600

Pask, Andrew J; Behringer, Richard R; Renfree, Marilyn B

2008-05-21

26

Resurrection of DNA Function In Vivo from an Extinct Genome  

PubMed Central

There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

Pask, Andrew J.; Behringer, Richard R.; Renfree, Marilyn B.

2008-01-01

27

Effects of Aspirin and Hypothermia on Platelet Function in Vivo.  

National Technical Information Service (NTIS)

Hypothermia, aspirin, and cardiopulmonary bypass can each induce a platelet function defect, but it is not known if the effects of aspirin and hypothermia are additive in this regard. To address this question in humans in vivo, the forearm skin temperatur...

A. D. Michelson M. R. Barnard S. F. Khuri M. J. Rohrer H. MacGregor

1997-01-01

28

Human myocardial ATP content and in vivo contractile function  

Microsoft Academic Search

The study was designed to characterize the relationship between the metabolise content of human cardiac muscle and in vivo cardiac function. ATP, total adenine nucleotides, and NAD were quantified in human myocardial biopsies using high performance liquid chromatography. Right ventricular endomyocardial biopsies were obtained from 43 patients with dilated cardiomyopathy, 6 with restrictive cardiomyopathy, 10 with normal systolic and diastolic

Randall C. Starling; Donald F. Hammer; Ruth A. Altschuld

1998-01-01

29

In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes1[W][OA  

PubMed Central

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx5HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.

Reumann, Sigrun; Quan, Sheng; Aung, Kyaw; Yang, Pingfang; Manandhar-Shrestha, Kalpana; Holbrook, Danielle; Linka, Nicole; Switzenberg, Robert; Wilkerson, Curtis G.; Weber, Andreas P.M.; Olsen, Laura J.; Hu, Jianping

2009-01-01

30

In vivo and in vitro analysis of the human tissue-type plasminogen activator gene promoter in neuroblastomal cell lines: evidence for a functional upstream kappaB element.  

PubMed

Besides its well-established role in wound healing and fibrinolysis, tissue-type plasminogen activator (t-PA) has been shown to contribute to cognitive processes and memory formation within the central nervous system, and to promote glutamate receptor-mediated excitotoxicity. The t-PA gene is expressed and regulated in neuronal cells but the regulatory transcriptional processes directing this expression are still poorly characterized. We have used DNase I-hypersensitivity mapping and in vivo foot printing to identify putative regulatory elements and transcription factor binding sites in two human neuroblastomal (KELLY and SK-N-SH) and one human glioblastomal (SNB-19) cell lines. Hypersensitive sites were found in the proximal promoter region of all cell lines, and within the first exon for KELLY and SNB-19 cells. Mapping of methylation-protected residues in vivo detected a cluster of protected residues corresponding to a cAMP response element (CRE) and Sp1 sites in the proximal promoter previously shown to be essential for basal expression in other cell types. Protected residues were also found at other sites, notably a kappaB element at position bp -3081 to -3072 that was partly protected in KELLY and SNB-19 cells. Analysis of transfected reporter constructs in KELLY and SNB-19 cells confirmed that this particular element is functionally significant in the transactivation of the t-PA promoter in both cell types. This study defines, by in vivo and in vitro methods, a previously undescribed kappaB site in the t-PA gene promoter that influences t-PA expression in neuronal cells. PMID:15869598

Lux, W; Klobeck, H-G; Daniel, P B; Costa, M; Medcalf, R L; Schleuning, W-D

2005-05-01

31

Detection of Tight Junction Barrier Function In Vivo by Biotin  

PubMed Central

Tight junctions (TJs) are the most apical component of the junctional complexes in mammalian epithelial cells and form selective paracellular barriers restricting the passage of solutes and ions across the epithelial sheets. Claudins, a TJ integral membrane protein family, play a critical role in regulating paracellular barrier permeability. In the in vitro cell culture system, transepithelial electrical resistance (TER) measurement and the flux of radioisotope or fluorescent labeled molecules with different sizes have been widely used to determine the TJ barrier function. In the in vivo system, the tracer molecule Sulfo-NHS-Biotin was initially used in Xenopus embryos system and subsequently was successfully applied to a number of animal tissues in situ and in different organisms under the experimental conditions to examine the functional integrity of TJs by several laboratories. In this chapter, we will describe the detailed procedures of applying biotin as a paracellular tracer molecule to different in vivo systems to assay TJ barrier function.

Ding, Lei; Zhang, Yuguo; Tatum, Rodney; Chen, Yan-Hua

2011-01-01

32

Context-dependent function of "GATA switch" sites in vivo.  

PubMed

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo. PMID:21398579

Snow, Jonathan W; Trowbridge, Jennifer J; Johnson, Kirby D; Fujiwara, Tohru; Emambokus, Nikla E; Grass, Jeffrey A; Orkin, Stuart H; Bresnick, Emery H

2011-03-11

33

Functional role of TRPC proteins in vivo: lessons from TRPC-deficient mouse models  

Microsoft Academic Search

In order to elucidate the functional role of TRPC genes, in vivo, the targeted inactivation of these genes in mice is an invaluable technique. In this review, we summarize the currently available results on the phenotype of TRPC-deficient mouse lines. The analysis of mice with targeted deletion in three TRPC genes demonstrates that these proteins represent essential constituents of agonist-activated

M. Freichel; R. Vennekens; J. Olausson; M. Hoffmann; C. Müller; S. Stolz; J. Scheunemann; P. Weißgerber; V. Flockerzi

2004-01-01

34

In Vivo Analysis of Conserved C. elegans Tomosyn Domains  

PubMed Central

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two ?-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.

Shtessel, Ludmila; Ahmed, Shawn; Richmond, Janet E.

2011-01-01

35

Dendritic spines: from structure to in vivo function  

PubMed Central

Dendritic spines arise as small protrusions from the dendritic shaft of various types of neuron and receive inputs from excitatory axons. Ever since dendritic spines were first described in the nineteenth century, questions about their function have spawned many hypotheses. In this review, we introduce understanding of the structural and biochemical properties of dendritic spines with emphasis on components studied with imaging methods. We then explore advances in in vivo imaging methods that are allowing spine activity to be studied in living tissue, from super-resolution techniques to calcium imaging. Finally, we review studies on spine structure and function in vivo. These new results shed light on the development, integration properties and plasticity of spines.

Rochefort, Nathalie L; Konnerth, Arthur

2012-01-01

36

Functional Analysis of CD28/B7 and CD40/CD40L Costimulation During the in vivo Type 2 Immune Response.  

National Technical Information Service (NTIS)

Cytokine production and other effector functions of CD4+ T helper (Th) cells are crucial for the initiation of a primary immune response. The activation of naive CD4+ Th cells requires two signals delivered from antigen presenting cells (APCs). The engage...

P. Lu

1995-01-01

37

Functionalized Magnetic Nanoparticles as an In Vivo Delivery System  

NASA Astrophysics Data System (ADS)

We developed extremely small functionalized magnetic nanoparticles (MNPs) for use as an in vivo delivery system for pharmaceuticals and biomolecules. We functionalized the MNPs (d = 3 nm) by silanization of amino groups on the particles with (3-aminopropyl)triethoxysilane for subsequent cross-linking with pharmaceuticals and biomolecules. The MNPs were successfully introduced into living cells without any further modification, such as the use of cationic residues, to enhance endocytic internalization. The particles could be incorporated into the subcutaneous tissue of a mouse’s ear through the skin of the ear and could be localized by application of an external magnetic field.

Taira, Shu; Moritake, Shinji; Hatanaka, Takahiro; Ichiyanagi, Yuko; Setou, Mitsutoshi

38

Physiological functions of protein kinase D in vivo.  

PubMed

The cellular functions of the serine/threonine protein kinase D (PKD) have been extensively studied within the last decade and distinct roles such as fission of vesicles at the Golgi compartment, coordination of cell migration and invasion, and regulation of gene transcription have been correlated with this kinase family. Here, we highlight the current state of in vivo studies on PKD function with a focus on animal models and discuss the molecular basis of the observed phenotypic characteristics associated with this kinase family. PMID:23288632

Ellwanger, Kornelia; Hausser, Angelika

2013-01-03

39

GmFtsH9 expression correlates with in vivo photosystem II function: chlorophyll a fluorescence transient analysis and eQTL mapping in soybean  

Microsoft Academic Search

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease involved in diverse biological functions.\\u000a There are 12 FtsH proteins in Arabidopsis, among which AtFtsH2 plays an important role in regulating the turnover of photosystem\\u000a II (PSII) reaction center D1 protein and the development of the photosynthetic apparatus. Here, we have identified 11 FtsH\\u000a genes in the soybean genome by a

Zhitong Yin; Fanfan Meng; Haina Song; Xiaolin Wang; Maoni Chao; Guozheng Zhang; Xiaoming Xu; Dexiang Deng; Deyue Yu

40

Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice.  

PubMed

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe's disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean ?-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA(-/-)) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity. PMID:23907679

Martiniuk, Frank; Reggi, Serena; Tchou-Wong, Kam-Meng; Rom, William N; Busconi, Matteo; Fogher, Corrado

2013-08-02

41

Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo.  

PubMed

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats. PMID:23063685

Harms, Floor A; Voorbeijtel, Wilhelmina J; Bodmer, Sander I A; Raat, Nicolaas J H; Mik, Egbert G

2012-10-12

42

Posterior lymph heart function in two species of anurans: analysis based on both in vivo pressure-volume relationships by conductance manometry and ultrasound  

PubMed Central

Rhinella marina and Lithobates catesbeianus have known differences in the capacity to mobilize lymph to stabilize blood volume following dehydration and hemorrhage. The purpose of these experiments was to assess whether there are interspecific differences in basic lymph heart functions. The end diastolic volumes of posterior lymph hearts averaged 10.8 ?l kg–1 in R. marina and 7.9–10.8 ?l kg–1 in L. catesbeianus by conductance manometry, and 9–32 ?l kg–1 in R. marina by ultasound techniques, which correlated with body mass. Stroke volumes were approximately 20% of end diastolic volumes in both species. Peak systolic pressures and stroke work were correlated with the index of contractility (dP/dtmax) in both species. Stroke volume was correlated to stroke work but not peak systolic pressure, end diastolic volume or end diastolic pressure indicating the preload variables do not seem to determine stroke volume as would be predicted from Starling considerations of the blood heart. Renal portal elastance (end systolic pressure/stroke volume) an afterload index did not differ interspecifically, and was equivalent to values for systemic flow indices from mice of equivalent ventricular volume. These data, taken together with predictions derived from mammalian models on the effect of high resistance indicate afterload (renal portal pressure), may be important determinants of posterior lymph heart stroke volume. The shape of the pressure–volume loop is different from an idealized version previously reported, and is influenced by end diastolic volume. Our data indicate that increasing end diastolic pressure and volume can influence the loop shape but not the stroke volume. This indicates that lymph hearts do not behave in a Starling Law manner with increased preload volume.

Crossley, Dane A.; Hillman, Stanley S.

2010-01-01

43

Identification and In Vivo Functional Analysis by Gene Disruption of ctnA, an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus? †  

PubMed Central

Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5?-flanking region and one ORF (orf5) in the 3?-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.

Shimizu, Takeo; Kinoshita, Hiroshi; Nihira, Takuya

2007-01-01

44

Neurovascular coupling: in vivo optical techniques for functional brain imaging.  

PubMed

Optical imaging techniques reflect different biochemical processes in the brain, which is closely related with neural activity. Scientists and clinicians employ a variety of optical imaging technologies to visualize and study the relationship between neurons, glial cells and blood vessels. In this paper, we present an overview of the current optical approaches used for the in vivo imaging of neurovascular coupling events in small animal models. These techniques include 2-photon microscopy, laser speckle contrast imaging (LSCI), voltage-sensitive dye imaging (VSDi), functional photoacoustic microscopy (fPAM), functional near-infrared spectroscopy imaging (fNIRS) and multimodal imaging techniques. The basic principles of each technique are described in detail, followed by examples of current applications from cutting-edge studies of cerebral neurovascular coupling functions and metabolic. Moreover, we provide a glimpse of the possible ways in which these techniques might be translated to human studies for clinical investigations of pathophysiology and disease. In vivo optical imaging techniques continue to expand and evolve, allowing us to discover fundamental basis of neurovascular coupling roles in cerebral physiology and pathophysiology. PMID:23631798

Liao, Lun-De; Tsytsarev, Vassiliy; Delgado-Martínez, Ignacio; Li, Meng-Lin; Erzurumlu, Reha; Vipin, Ashwati; Orellana, Josue; Lin, Yan-Ren; Lai, Hsin-Yi; Chen, You-Yin; Thakor, Nitish V

2013-04-30

45

Imaging visual cortical structure and function in vivo.  

PubMed

The recent advent of in vivo two-photon microscopy has allowed the repeat imaging of cortical structures at microscopic resolution within intact brains. Recent data obtained using this imaging technique shows that dendritic spines, the postsynaptic sites of the majority of excitatory synapses in the central nervous system (CNS), rapidly remodel in response to changes in the visual environment. We combined two-photon microscopy of dendritic segments with intrinsic signal imaging of visual cortical responses in the developing ferret visual cortex, and showed that when one eye was deprived during the developmental critical period for ocular dominance plasticity, both dendritic spines and visual responses to the deprived eye were rapidly altered. A brief period of recovery where the eye was re-opened resulted in a return to pre-deprivation levels for both responses and dendritic spine density, showing that structural and functional changes are linked even at very rapid timescales. Additionally, two-photon microscopy can assay other functional and structural aspects of visual cortical function which I will review. Lastly, I will compare this technique to other imaging modalities available for assessment of the visual cortex in vivo. PMID:23733120

Majewska, Ania K

46

Techniques for the in vivo assessment of cardio-renal function in zebrafish (Danio rerio) larvae  

PubMed Central

Zebrafish, a well-established vertebrate model, offer unique advantages for assessing renal function and physiology. Assays determining renal glomerular function based on cardiovascular erythrocyte flow and reduction of injected FITC-inulin were developed, each validated using the nephrotoxin gentamicin. Bland–Atlman analysis showed a strong association between measurements of the rate of inulin excretion and that of fluorescent reduction from the arterial vasculature. Reduced renal clearance of inulin, resulting from gentamicin or NaCl loading, was concurrent with reduced erythrocyte velocity, and yolk sac and pericardium oedema. These techniques, assessing pronephric function, highlight the potential for in vivo physiological study in this genetically tractable model.

Rider, Sebastien A; Tucker, Carl S; del-Pozo, Jorge; Rose, Kirsten N; MacRae, Calum A; Bailey, Matthew A; Mullins, John J

2012-01-01

47

In-vivo plasma-mediated ablation as a function of laser pulse width  

NASA Astrophysics Data System (ADS)

We evaluated in vivo wound healing responses to plasma- mediated ablation in skin as a function of laser pulsewidth and energy. Experiments utilized a regeneratively amplified Ti:Sapphire laser operating at 800 nm with pulsewidths varied from 7 ns to 100 fs. Skin incisions were created in mice by tightly focusing the laser beam on the tissue surface. Incisions of equal depth were compared at time points ranging from 6 hours to 3 weeks using standard histologic methods. Incision depth was proportional to pulse energy at each pulsewidth. Fluence threshold dependence on laser pulsewidth agreed with those predicted by ex vivo testing. Histologic analysis revealed minimal adjacent tissue damage at pulsewidths less than a few picoseconds and energies near the fluence threshold. Longer pulsewidths and higher fluence levels were associated with more significant collateral effects. These in vivo results suggest collateral tissue damage and secondary effects may be minimized by controlling laser pulsewidth and energy.

Liu, Xinbing; Tien, An Xien; Juhasz, Tibor; Irish, Barbara; Elner, Victor; Kurtz, Ron M.

1997-06-01

48

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function*  

PubMed Central

The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of “deviant” Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function.

Boyd, Jeffrey M.; Sondelski, Jamie L.; Downs, Diana M.

2009-01-01

49

Multilevel functional clustering analysis.  

PubMed

In this article, we investigate clustering methods for multilevel functional data, which consist of repeated random functions observed for a large number of units (e.g., genes) at multiple subunits (e.g., bacteria types). To describe the within- and between variability induced by the hierarchical structure in the data, we take a multilevel functional principal component analysis (MFPCA) approach. We develop and compare a hard clustering method applied to the scores derived from the MFPCA and a soft clustering method using an MFPCA decomposition. In a simulation study, we assess the estimation accuracy of the clustering membership and the cluster patterns under a series of settings: small versus moderate number of time points; various noise levels; and varying number of subunits per unit. We demonstrate the applicability of the clustering analysis to a real data set consisting of expression profiles from genes activated by immunity system cells. Prevalent response patterns are identified by clustering the expression profiles using our multilevel clustering analysis. PMID:22313290

Serban, Nicoleta; Jiang, Huijing

2012-02-07

50

Use of photoproteins for in-vivo functional imaging  

NASA Astrophysics Data System (ADS)

The relative opacity of mammalian tissue permits the transmission of light from internal biological light sources in small laboratory animals. As such internally expressed bioluminescence can be detected externally revealing spatiotemporal information about tagged biological functions. Enzymes that emit light, photoproteins, have been characterized photoproteins have been used as reporters in a variety of in vitro and ex vivo assays and are now being employed as sources of internal biological light that can be eternally monitored in living animals. Using this approach, spatiotemporal changes in patterns of gene expression, infectious disease and tumor cell growth can be revealed in real time. Monitoring light emissions from internal sources provides a powerful method for cellular and molecular analyses in living animals. This approach is particularly well suited for the evaluation of potential therapeutics including the efficacy of novel DNA-based therapies and vaccines.

Contag, Christopher H.

1999-07-01

51

In vivo minimally invasive interstitial multi-functional microendoscopy  

NASA Astrophysics Data System (ADS)

Developing minimally invasive methodologies for imaging of internal organs is an emerging field in the biomedical examination research. This paper introduces a new multi-functional microendoscope device capable of imaging of internal organs with a minimal invasive intervention. In addition, the developed microendoscope can also be employed as a monitoring device for measuring local hemoglobin concentration in blood stream when administrated into a blood artery. The microendoscope device has a total external diameter of only 200 ?m and can provide high imaging resolution capability of more than 5,000 pixels. The device can detect features with a spatial resolution of less than 1 ?m. The microendoscope has been tested both in-vitro as well as in-vivo in rats presenting a promising and powerful tool as a high resolution and minimally invasive imaging facility suitable for previously unreachable clinical modalities.

Shahmoon, Asaf; Aharon, Shiran; Kruchik, Oded; Hohmann, Martin; Slovin, Hamutal; Douplik, Alexandre; Zalevsky, Zeev

2013-05-01

52

Emerging In Vivo Analyses of Cell Function Using Fluorescence Imaging?  

PubMed Central

Understanding how cells of all types sense external and internal signals and how these signals are processed to yield particular responses is a major goal of biology. Genetically encoded fluorescent proteins (FPs) and fluorescent sensors are playing an important role in achieving this comprehensive knowledge base of cell function. Providing high sensitivity and immense versatility while being minimally perturbing to a biological specimen, the probes can be used in different microscopy techniques to visualize cellular processes on many spatial scales. Three review articles in this volume discuss recent advances in probe design and applications. These developments help expand the range of biochemical processes in living systems suitable for study. They provide researchers with exciting new tools to explore how cellular processes are organized and their activity regulated in vivo.

Lippincott-Schwartz, Jennifer

2013-01-01

53

Cadmium by in vivo neutron activation analysis  

Microsoft Academic Search

\\u000a Résumé  On peut déterminer la présence de cadmium chez l'homme ŕ l'aide de technique de l'analyse par activation neutronique in vivo.\\u000a La capture des neutrons thermiques par113Cd conduit ŕ une émission ? prompt qui peut ętre détectée au moyen d'un semi-conducteur convenable. On a réalisé une installation\\u000a ŕ l'Université de Birmingham pour produire au moyen d'un cyclotron un faisceau de neutrons

H. C. Biggin; N. S. Chen; K. V. Ettinger; J. H. Fremlin; W. D. Morgan; R. Nowotny; M. J. Chamberlain; T. C. Harvey

1974-01-01

54

Function and specificity of synthetic Hox transcription factors in vivo  

PubMed Central

Homeotic (Hox) genes encode transcription factors that confer segmental identity along the anteroposterior axis of the embryo. However the molecular mechanisms underlying Hox-mediated transcription and the differential requirements for specificity in the regulation of the vast number of Hox-target genes remain ill-defined. Here we show that synthetic Sex combs reduced (Scr) genes that encode the Scr C terminus containing the homedomain (HD) and YPWM motif (Scr-HD) are functional in vivo. Synthetic Scr-HD peptides can induce ectopic salivary glands in the embryo and homeotic transformations in the adult fly, act as transcriptional activators and repressors during development, and participate in protein-protein interactions. Their transformation capacity was found to be enhanced over their full-length counterpart and mutations known to transform the full-length protein into constitutively active or inactive variants behaved accordingly in the synthetic peptides. Our results show that synthetic Scr-HD genes are sufficient for homeotic function in Drosophila and suggest that the N terminus of Scr has a role in transcriptional potency, rather than specificity. We also demonstrate that synthetic peptides behave largely in a predictable way, by exhibiting Scr-specific phenotypes throughout development, which makes them an important tool for synthetic biology.

Papadopoulos, Dimitrios K.; Vukojevic, Vladana; Adachi, Yoshitsugu; Terenius, Lars; Rigler, Rudolf; Gehring, Walter J.

2010-01-01

55

A Primer on Functional Analysis  

ERIC Educational Resources Information Center

|This article presents principles and basic steps for practitioners to complete a functional analysis of client behavior. The emphasis is on application of functional analysis to adult mental health clients. The article includes a detailed flow chart containing all major functional diagnoses and behavioral interventions, with functional assessment…

Yoman, Jerome

2008-01-01

56

DHA-enriched fish oil targets B cell lipid microdomains and enhances ex vivo and in vivo B cell function.  

PubMed

DHA is a n-3 LCPUFA in fish oil that generally suppresses T lymphocyte function. However, the effect of fish oil on B cell function remains relatively understudied. Given the important role of B cells in gut immunity and increasing human fish oil supplementation, we sought to determine whether DFO leads to enhanced B cell activation in the SMAD-/- colitis-prone mouse model, similar to that observed with C57BL/6 mice. This study tested the hypothesis that DHA from fish oil is incorporated into the B cell membrane to alter lipid microdomain clustering and enhance B cell function. Purified, splenic B cells from DFO-fed mice displayed increased DHA levels and diminished GM1 microdomain clustering. DFO enhanced LPS-induced B cell secretion of IL-6 and TNF-? and increased CD40 expression ex vivo compared with CON. Despite increased MHCII expression in the unstimulated ex vivo B cells from DFO-fed mice, we observed no difference in ex vivo OVA-FITC uptake in B cells from DFO or CON mice. In vivo, DFO increased lymphoid tissue B cell populations and surface markers of activation compared with CON. Finally, we investigated whether these ex vivo and in vivo observations were consistent with systemic changes. Indeed, DFO-fed mice had significantly higher plasma IL-5, IL-13, and IL-9 (Th2-biasing cytokines) and cecal IgA compared with CON. These results support the hypothesis and an emerging concept that fish oil enhances B cell function in vivo. PMID:23180828

Gurzell, Eric A; Teague, Heather; Harris, Mitchel; Clinthorne, Jonathan; Shaikh, Saame Raza; Fenton, Jenifer I

2012-11-24

57

Inflammation Modulates Human HDL Composition and Function in vivo  

PubMed Central

Objectives Inflammation may directly impair HDL functions, in particular reverse cholesterol transport (RCT), but limited data support this concept in humans. Methods and Results We employed low-dose human endotoxemia to assess the effects of inflammation on HDL and RCT-related parameters in vivo. Endotoxemia induced remodelling of HDL with depletion of pre-?1a HDL particles determined by 2-D gel electrophoresis (-32.2 ± 9.3% at 24h, p<0.05) as well as small (-23.0 ± 5.1%, p<0.01, at 24h) and medium (-57.6 ± 8.0% at 16h, p<0.001) HDL estimated by nuclear magnetic resonance (NMR). This was associated with induction of class II secretory phospholipase A2 (~36 fold increase) and suppression of lecithin:cholesterol acyltransferase activity (-20.8 ± 3.4% at 24h, p<0.01) and cholesterol ester transfer protein mass (-22.2 ± 6.8% at 24h, p<0.001). The HDL fraction, isolated following endotoxemia, had reduced capacity to efflux cholesterol in vitro from SR-BI and ABCA1, but not ABCG1 transporter cell models. Conclusions These data support the concept that “atherogenic-HDL dysfunction” and impaired RCT occur in human inflammatory syndromes, largely independent of changes in plasma HDL-C and ApoA-I levels.

de la Llera Moya, Margarita; McGillicuddy, Fiona C; Hinkle, Christine C; Byrne, Michael; Joshi, Michelle R; Nguyen, Vihn; Tabita-Martinez, Jennifer; Wolfe, Megan L; Badellino, Karen; Pruscino, Leticia; Mehta, Nehal N; Asztalos, Bela F; Reilly, Muredach P

2012-01-01

58

Transplanted CNS stem cells form functional synapses in vivo.  

PubMed

An understanding of developmental mechanisms and new cell therapies can be achieved by transplantation into the nervous system. Multipotential stem cells have been isolated from the foetal and adult central nervous system (CNS). Immortalized and primary precursor cells integrate into the developing brain generating both neurons and glia as defined by immunological and morphological criteria. Here we show for the first time that in vitro-expanded CNS precursors, upon transplantation into the brains of rats, form electrically active and functionally connected neurons. These neurons exhibit spontaneous and evoked postsynaptic events and respond to focal glutamate application. Donor cells were grafted into the foetal hippocampus, and the amplitude and frequency of spontaneous synaptic events were monitored in the grafted cells in area CA1 for the first month of postnatal life. The formation of synapses onto grafted neurons indicates that grafted CNS stem cells can be used to study synaptic development in vivo and has important implications for clinical cell replacement therapies. PMID:10792447

Auerbach, J M; Eiden, M V; McKay, R D

2000-05-01

59

A comprehensive ex vivo functional analysis of human NKT cells reveals production of MIP1-? and MIP1-?, a lack of IL-17, and a Th1-bias in males.  

PubMed

NKT cells contribute to the modulation of immune responses and are believed to be important in the pathogenesis of autoimmune and infectious diseases, as well as cancer. Variations in the composite NKT cytokine response may determine individual disease susceptibility or severity. Due to low frequencies in peripheral blood, knowledge of the breadth of ex vivo human NKT cell functions has been limited. To bridge this gap, we studied highly purified NKT cells from PBMC of healthy donors and assessed the production of 27 effector functions using sensitive Elispot and multiplex bead assays. We found the ex vivo human NKT cell response is predominantly comprised of the chemokines MIP1-?, and MIP1-? as well as the Th1 cytokines IFN-? and TNF-?. Although lower in magnitude, there was also significant production of IL-2, IL-4, and perforin after mitogen stimulation. Surprisingly, little/no IL-5, IL-6, IL-10, or IL-13 was detected, and no subjects' NKT cells produced IL-17. Comparison of the NKT functional profiles between age-matched male and female subjects revealed similar IL-4 responses, but higher frequencies of cells producing IFN-? and MIP1-?, from males. There were no gender differences in the circulating NKT subset distribution. These findings implicate chemokines as a major mechanism by which NKT cells control responses in humans. In addition, the panoply of Th2 and Th17 cytokine secretion by NKT cells from healthy donors may not be as pronounced as previously believed. NKT cells may therefore contribute to the gender bias found in many diseases. PMID:21082024

Snyder-Cappione, Jennifer E; Tincati, Camilla; Eccles-James, Ijeoma G; Cappione, Amedeo J; Ndhlovu, Lishomwa C; Koth, Laura L; Nixon, Douglas F

2010-11-03

60

Analysis of cortical flow models in vivo.  

PubMed

Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment of Xenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center in Xenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow. PMID:10930453

Benink, H A; Mandato, C A; Bement, W M

2000-08-01

61

Texture analysis of optical coherence tomography speckle for characterizing biological tissues in vivo.  

PubMed

We demonstrate a method for differentiating tissue disease states using the intrinsic texture properties of speckle in optical coherence tomography (OCT) images of normal and tumor tissues obtained in vivo. This approach fits a gamma distribution function to the nonlog-compressed OCT image intensities, thus allowing differentiation of normal and tumor tissues in an ME-180 human cervical cancer mouse xenograft model. Quantitative speckle intensity distribution analysis thus shows promise for identifying tissue pathologies, with potential for early cancer detection in vivo. PMID:23595458

Lindenmaier, Andras A; Conroy, Leigh; Farhat, Golnaz; DaCosta, Ralph S; Flueraru, Costel; Vitkin, I Alex

2013-04-15

62

Functional Group Analysis.  

ERIC Educational Resources Information Center

|Literature on analytical methods related to the functional groups of 17 chemical compounds is reviewed. These compounds include acids, acid azides, alcohols, aldehydes, ketones, amino acids, aromatic hydrocarbons, carbodiimides, carbohydrates, ethers, nitro compounds, nitrosamines, organometallic compounds, peroxides, phenols, silicon compounds,…

Smith, Walter T., Jr.; Patterson, John M.

1984-01-01

63

Identification and analysis of bacterial virulence genes in vivo.  

PubMed Central

Signature-tagged mutagenesis is a mutation-based screening method for the identification of virulence genes of microbial pathogens. Genes isolated by this approach fall into three classes: those with known biochemical function, those of suspected function and some whose functions cannot be predicted from database searches. A variety of in vitro and in vivo methods are available to elucidate the function of genes of the second and third classes. We describe the use of some of these approaches to study the function of the Salmonella pathogenicity island 2 type III secretion system of Salmonella typhimurium. This virulence determinant is required for intracellular survival. Secretion by this system is induced by an acidic pH, and its function may be to alter trafficking of the Salmonella-containing vacuole. Use of a temperature-sensitive non-replicating plasmid and competitive index tests with other genes show that in vivo phenotypes do not always correspond to those predicted from in vitro studies.

Unsworth, K E; Holden, D W

2000-01-01

64

In vivo function of the craniofacial haft: The interorbital ?pillar?  

Microsoft Academic Search

The craniofacial haft resists forces gener- ated in the face during feeding, but the importance of these forces for the form of the craniofacial haft remains to be determined. In vivo bone strain data were recorded from the medial orbital wall in an owl monkey (Aotus), rhesus macaques (Macaca mulatta), and a galago (Otole- mur) during feeding. These data were

Callum F. Ross

2001-01-01

65

Function Analysis and Decomposistion using Function Analysis Systems Technique  

SciTech Connect

The "Father of Value Analysis", Lawrence D. Miles, was a design engineer for General Electric in Schenectady, New York. Miles developed the concept of function analysis to address difficulties in satisfying the requirements to fill shortages of high demand manufactured parts and electrical components during World War II. His concept of function analysis was further developed in the 1960s by Charles W. Bytheway, a design engineer at Sperry Univac in Salt Lake City, Utah. Charles Bytheway extended Mile's function analysis concepts and introduced the methodology called Function Analysis Systems Technique (FAST) to the Society of American Value Engineers (SAVE) at their International Convention in 1965 (Bytheway 1965). FAST uses intuitive logic to decompose a high level, or objective function into secondary and lower level functions that are displayed in a logic diagram called a FAST model. Other techniques can then be applied to allocate functions to components, individuals, processes, or other entities that accomplish the functions. FAST is best applied in a team setting and proves to be an effective methodology for functional decomposition, allocation, and alternative development.

Wixson, James Robert

1999-06-01

66

Function Analysis and Decomposistion using Function Analysis Systems Technique  

SciTech Connect

The "Father of Value Analysis", Lawrence D. Miles, was a design engineer for General Electric in Schenectady, New York. Miles developed the concept of function analysis to address difficulties in satisfying the requirements to fill shortages of high demand manufactured parts and electrical components during World War II. His concept of function analysis was further developed in the 1960s by Charles W. Bytheway, a design engineer at Sperry Univac in Salt Lake City, Utah. Charles Bytheway extended Mile's function analysis concepts and introduced the methodology called Function Analysis Systems Techniques (FAST) to the Society of American Value Engineers (SAVE) at their International Convention in 1965 (Bytheway 1965). FAST uses intuitive logic to decompose a high level, or objective function into secondary and lower level functions that are displayed in a logic diagram called a FAST model. Other techniques can then be applied to allocate functions to components, individuals, processes, or other entities that accomplish the functions. FAST is best applied in a team setting and proves to be an effective methodology for functional decomposition, allocation, and alternative development.

J. R. Wixson

1999-06-01

67

In Vivo Application of Optogenetics for Neural Circuit Analysis  

PubMed Central

Optogenetics combines optical and genetic methods to rapidly and reversibly control neural activities or other cellular functions. Using genetic methods, specific cells or anatomical pathways can be sensitized to light through exogenous expression of microbial light activated opsin proteins. Using optical methods, opsin expressing cells can be rapidly and reversibly controlled by pulses of light of specific wavelength. With the high spatial temporal precision, optogenetic tools have enabled new ways to probe the causal role of specific cells in neural computation and behavior. Here, we overview the current state of the technology, and provide a brief introduction to the practical considerations in applying optogenetics in vivo to analyze neural circuit functions.

2012-01-01

68

Ena/VASP is required for endothelial barrier function in vivo  

PubMed Central

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia.

Furman, Craig; Sieminski, Alisha L.; Kwiatkowski, Adam V.; Rubinson, Douglas A.; Vasile, Eliza; Bronson, Roderick T.; Fassler, Reinhard; Gertler, Frank B.

2007-01-01

69

Selection of antibodies for intracellular function using a two-hybrid in vivo system  

PubMed Central

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Visintin, Michela; Tse, Eric; Axelson, Hakan; Rabbitts, Terence H.; Cattaneo, Antonino

1999-01-01

70

Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo  

PubMed Central

Background Inhalation of diesel exhaust impairs vascular function in man, by a mechanism that has yet to be fully established. We hypothesised that pulmonary exposure to diesel exhaust particles (DEP) would cause endothelial dysfunction in rats as a consequence of pulmonary and systemic inflammation. Methods Wistar rats were exposed to DEP (0.5 mg) or saline vehicle by intratracheal instillation and hind-limb blood flow, blood pressure and heart rate were monitored in situ 6 or 24 h after exposure. Vascular function was tested by administration of the endothelium-dependent vasodilator acetylcholine (ACh) and the endothelium-independent vasodilator sodium nitroprusside (SNP) in vivo and ex vivo in isolated rings of thoracic aorta, femoral and mesenteric artery from DEP exposed rats. Bronchoalveolar lavage fluid (BALF) and blood plasma were collected to assess pulmonary (cell differentials, protein levels & interleukin-6 (IL-6)) and systemic (IL-6), tumour necrosis factor alpha (TNF?) and C-reactive protein (CRP)) inflammation, respectively. Results DEP instillation increased cell counts, total protein and IL-6 in BALF 6 h after exposure, while levels of IL-6 and TNF? were only raised in blood 24 h after DEP exposure. DEP had no effect on the increased hind-limb blood flow induced by ACh in vivo at 6 or 24 h. However, responses to SNP were impaired at both time points. In contrast, ex vivo responses to ACh and SNP were unaltered in arteries isolated from rats exposed to DEP. Conclusions Exposure of rats to DEP induces both pulmonary and systemic inflammation, but does not modify endothelium-dependent vasodilatation. Other mechanisms in vivo limit dilator responses to SNP and these require further investigation.

2012-01-01

71

Nitric Oxide Effects on the Function of Aged Cells Ex Vivo and In Vivo  

PubMed Central

Background Angiogenesis is impaired in most aged tissues. Accordingly, there is great interest in interventions that improve the ability of aged cells to undergo blood vessel formation and subsequent tissue repair. Materials and Methods Nitric oxide (NO), a mediator proposed to enhance angiogenesis, was administered (as the precursor SNAP, S-nitroso-N-acetylpenicillamine) to aortic ring explants from aged mice and to aged mice in two separate in vivo experiments; a PVA sponge implant model of angiogenesis and full thickness excisional dermal wounds. Results SNAP inhibited angiogenesis from the mouse aortic ring explants. However, there was a trend toward increased blood vessel formation in the sponges from the aged mice treated with SNAP. SNAP did not detectably enhance dermal wound healing or angiogenesis, but it significantly inhibited epidermal closure. Conclusion These data underscore the complexity of using a single agent, even one with multiple mechanisms such as NO, to improve a clinical outcome such as angiogenesis or wound repair in aged animals.

Reed, May J.; Eyman, Daniel; Karres, Nathan

2009-01-01

72

In vitro gene regulatory networks predict in vivo function of liver  

PubMed Central

Background Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. Results Here we use the nitroaromatic 2,4,6-trinitrotoluene (TNT) as a model chemical to compare and determine how we might extrapolate from in vitro data to in vivo effects. We found 341 transcripts differentially expressed in common among in vitro and in vivo assays in response to TNT. The major functional term corresponding to these transcripts was cell cycle. Similarly modulated common pathways were identified between in vitro and in vivo. Furthermore, we uncovered the conserved common transcriptional gene regulatory networks between in vitro and in vivo cellular liver systems that responded to TNT exposure, which mainly contain 2 subnetwork modules: PTTG1 and PIR centered networks. Interestingly, all 7 genes in the PTTG1 module were involved in cell cycle and downregulated by TNT both in vitro and in vivo. Conclusions The results of our investigation of TNT effects on gene expression in liver suggest that gene regulatory networks obtained from an in vitro system can predict in vivo function and mechanisms. Inhibiting PTTG1 and its targeted cell cyle related genes could be key machanism for TNT induced liver toxicity.

2010-01-01

73

Resurrection of DNA Function In Vivo from an Extinct Genome  

Microsoft Academic Search

There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from

Andrew J. Pask; Richard R. Behringer; Marilyn B. Renfree; Erik I. Svensson

2008-01-01

74

Mapping 3-D functional capillary geometry in rat skeletal muscle in vivo  

PubMed Central

We have developed a novel mapping software package to reconstruct microvascular networks in three dimensions (3-D) from in vivo video images for use in blood flow and O2 transport modeling. An intravital optical imaging system was used to collect video sequences of blood flow in microvessels at different depths in the tissue. Functional images of vessels were produced from the video sequences and were processed using automated edge tracking software to yield location and geometry data for construction of the 3-D network. The same video sequences were analyzed for hemodynamic and O2 saturation data from individual capillaries in the network. Simple user-driven commands allowed the connection of vessel segments at bifurcations, and semiautomated registration enabled the tracking of vessels across multiple focal planes and fields of view. The reconstructed networks can be rotated and manipulated in 3-D to verify vessel connections and continuity. Hemodynamic and O2 saturation measurements made in vivo can be indexed to corresponding vessels and visualized using colorized maps of the vascular geometry. Vessels in each reconstruction are saved as text-based files that can be easily imported into flow or O2 transport models with complete geometry, hemodynamic, and O2 transport conditions. The results of digital morphometric analysis of seven microvascular networks showed mean capillary diameters and overall capillary density consistent with previous findings using histology and corrosion cast techniques. The described mapping software is a valuable tool for the quantification of in vivo microvascular geometry, hemodynamics, and oxygenation, thus providing rich data sets for experiment-based computational models.

Milkovich, Stephanie; Goldman, Daniel; Ellis, Christopher G.

2012-01-01

75

In vivo analysis of the Hsp90 cochaperone Sti1 (p60).  

PubMed Central

Hsp90 interacts with Sti1 (p60) in lysates of yeast and vertebrate cells. Here we provide the first analysis of their interaction in vivo. Saccharomyces cerevisiae mutations that eliminate Sti1 or reduce intracellular concentrations of Hsp90 individually have little or no effect on growth at normal temperatures. However, when combined, the mutations greatly reduce or eliminate growth. Furthermore, overexpression of Sti1 has allele-specific effects on cells carrying various hsp90ts point mutations. These genetic interactions provide strong evidence that Hsp90 and Sti1 interact in vivo and that their functions are closely allied. Indeed, deletion of STI1 reduces the in vivo activity of the Hsp90 target protein, glucocorticoid receptor (GR). Mutations in GR that eliminate interaction with Hsp90 also eliminate the effects of the sti1 deletion. Examination of GR protein complexes in the sti1 deletion mutant reveals a selective increase in the concentration of GR-Ydj1 complexes, supporting previous hypotheses that Ydj1 functions at an early step in the maturation of GR and that Sti1 acts at an intermediate step. Deletion of STI1 also reduces the in vivo activity of another, unrelated Hsp90 target protein, v-Src. Our data indicate that Sti1 is a general factor in the maturation of Hsp90 target proteins and support earlier suggestions that Hsp90 matures even very different target proteins by a similar mechanism.

Chang, H C; Nathan, D F; Lindquist, S

1997-01-01

76

The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo.  

PubMed

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB(1) and CB(2) affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55(-/-) macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55(-/-) mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo. PMID:19805329

Whyte, Lauren S; Ryberg, Erik; Sims, Natalie A; Ridge, Susan A; Mackie, Ken; Greasley, Peter J; Ross, Ruth A; Rogers, Michael J

2009-09-03

77

The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo  

PubMed Central

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB1 and CB2 affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55?/? macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55?/? mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.

Whyte, Lauren S.; Ryberg, Erik; Sims, Natalie A.; Ridge, Susan A.; Mackie, Ken; Greasley, Peter J.; Ross, Ruth A.; Rogers, Michael J.

2009-01-01

78

Cyclin D1 Determines Mitochondrial Function In Vivo  

Microsoft Academic Search

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was

Toshiyuki Sakamaki; Mathew C. Casimiro; Xiaoming Ju; Andrew A. Quong; Sanjay Katiyar; Manran Liu; Xuanmao Jiao; Anping Li; Xueping Zhang; Yinan Lu; Chenguang Wang; Stephen Byers; Robert Nicholson; Todd Link; Melvin Shemluck; Jianguo Yang; Stanley T. Fricke; Phyllis M. Novikoff; Alexandros Papanikolaou; Andrew Arnold; Christopher Albanese; Richard Pestell

2006-01-01

79

Methods for the analysis of intestinal function.  

PubMed Central

The intestinal tract, an organ of considerable complexity, requires application of numerous techniques for analysis of its physiology and perturbations by toxicologic agents. This review describes the methodology of importance in analysis of the absorptive function of the intestine and the transit of intestinal contents. Methods for studying absorption are categorized according to the technique for administering the test substance such as inclusion in the diet or by gastric and intestinal placement and the method of quantitating the degree of absorption such as determining the appearance of a test substance in systemic fluids or its disappearance from its site of administration in the intestine. In vitro techniques which have no in vivo analogs, such as the use of the everted sac, are briefly described and their limitations emphasized. Procedures of importance in the clinical diagnosis of malabsorption or in the experimental analysis of absorptive function in man are included and distinguished from techniques used in animal models. In addition, methods for studying aspects of gastrointestinal motility, including the use of luminal markers and analysis of the contractile and electrical activity of intestinal smooth muscle, are reviewed. Images FIGURE 2.

Walsh, C T; Levine, R R

1979-01-01

80

In vivo functional imaging of human cone photoreceptors  

PubMed Central

We evaluate a novel non-invasive optical technique for observing fast physiological processes, in particular phototransduction, in single photoreceptor cells in the living human eye. The method takes advantage of the interference of multiple reflections within the outer segments (OS) of cones. This self-interference phenomenon is highly sensitive to phase changes such as those caused by variations in refractive index and scatter within the photoreceptor cell. A high-speed (192 Hz) flood-illumination retina camera equipped with adaptive optics (AO) is used to observe individual photoreceptors, and to monitor changes in their reflectance in response to visible stimuli (“scintillation”). AO and high frame rates are necessary for resolving individual cones and their fast temporal dynamics, respectively. Scintillation initiates within 5 to 10 ms after the onset of the stimulus flash, lasts 300 to 400 ms, is observed at visible and near-infrared (NIR) wavelengths, and is highly sensitive to the coherence length of the imaging light source. To our knowledge this is the first demonstration of in vivo optical imaging of the fast physiological processes that accompany phototransduction in individual photoreceptors.

Jonnal, Ravi S.; Rha, Jungtae; Zhang, Yan; Cense, Barry; Gao, Weihua; Miller, Donald T.

2009-01-01

81

In vivo functional imaging of human cone photoreceptors  

PubMed Central

We evaluate a novel non-invasive optical technique for observing fast physiological processes, in particular phototransduction, in single photoreceptor cells in the living human eye. The method takes advantage of the interference of multiple reflections within the outer segments (OS) of cones. This self-interference phenomenon is highly sensitive to phase changes such as those caused by variations in refractive index and scatter within the photoreceptor cell. A high-speed (192 Hz) flood-illumination retina camera equipped with adaptive optics (AO) is used to observe individual photoreceptors, and to monitor changes in their reflectance in response to visible stimuli (“scintillation”). AO and high frame rates are necessary for resolving individual cones and their fast temporal dynamics, respectively. Scintillation initiates within 5 to 10 ms after the onset of the stimulus flash, lasts 300 to 400 ms, is observed at visible and near-infrared (NIR) wavelengths, and is highly sensitive to the coherence length of the imaging light source. To our knowledge this is the first demonstration of in vivo optical imaging of the fast physiological processes that accompany phototransduction in individual photoreceptors.

Jonnal, Ravi S.; Rha, Jungtae; Zhang, Yan; Cense, Barry; Gao, Weihua; Miller, Donald T.

2008-01-01

82

In vivo and in vitro HeNe laser effects on phagocyte functions  

Microsoft Academic Search

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's

G. Ricevuti; A. Mazzone; C. Monaia; P. Fratino; R. Degiulio; R. Dell'acqua; G. Leonardi; A. Jucci; S. Sacchi

1989-01-01

83

The effects of flavanol-rich cocoa and aspirin on ex vivo platelet function  

Microsoft Academic Search

Background: Flavanols modulate platelet function in vitro, but less is known of their in vivo effects and how they compare to pharmacological platelet inhibitors. We investigated the effect of a flavanol-rich cocoa beverage (897 mg\\/ml) in combination with and in comparison to aspirin on platelet function and activation in healthy subjects. Methods and results: On separate test days in a

Debra A Pearson; Teresa G Paglieroni; Dietrich Rein; Ted Wun; Derek D Schramm; Janice F Wang; Roberta R Holt; Robert Gosselin; Harold H Schmitz; Carl L Keen

2002-01-01

84

Evaluation of effector cell fate and function by in vivo bioluminescence imaging  

Microsoft Academic Search

The effector functions of immune cells have typically been examined using assays that require sampling of tissues or cells to reveal specific aspects of an immune response (e.g., antigen-specificity, cytokine expression or killing of target cells). The outcome of an immune response in vivo, however, is not solely determined by a single effector function of a specific cell population, but

Matthias Edinger; Petra Hoffmann; Christopher H Contag; Robert S Negrin

2003-01-01

85

In vivo mapping of functional connectivity in neurotransmitter systems using pharmacological MRI.  

PubMed

Pharmacological MRI (phMRI) methods map the hemodynamic response to drug challenge as a surrogate for changes in neuronal activity. However, the central effects of drugs can be complex and include activity at the primary site of action, downstream effects in other brain regions and direct effects on vasculature and neurovascular coupling. Univariate analysis, normally applied to phMRI data, does not discriminate between these effects, and can result in anatomically non-specific activation patterns. We analysed inter-subject correlations in the amplitude of the slow phMRI response to map functionally connected brain regions recruited in response to pharmacological challenge. Application of D-amphetamine and fluoxetine revealed well-defined functional structure underlying the widespread signal changes detected via standard methods. Correlated responses were found to delineate key neurotransmitter pathways selectively targeted by these drugs, corroborating a tight correspondence between the phMRI response and changes in neurotransmitter systems specific to the pharmacological action. In vivo mapping of correlated responses in this way greatly extends the range of information available from phMRI studies and provides a new window into the function of neurotransmitter systems in the active state. This approach may provide new important insights regarding the central systems underlying pharmacological action. PMID:17188903

Schwarz, Adam J; Gozzi, Alessandro; Reese, Torsten; Bifone, Angelo

2006-12-26

86

Functional Analysis Proofs of Some Theorems in Function Theory.  

National Technical Information Service (NTIS)

Functional analysis proofs of three theorems in functions theory are given. The first theorem is Runge's theorem on approximation by rational functions. The second is the familiar theorem that there exists an analytic function that interpolates arbitrary ...

L. A. Rubel B. A. Taylor

1969-01-01

87

Exposure-in-vivo containing interventions to improve work functioning of workers with anxiety disorder: a systematic review  

PubMed Central

Background Anxiety disorders are associated with functional disability, sickness absence, and decreased productivity. Effective treatments of anxiety disorders can result in remission of symptoms. However the effects on work related outcomes are largely unknown. Exposure in vivo is potentially well fit to improve work-related outcomes. This study systematically reviews the effectiveness of exposure-in-vivo containing interventions in reducing work-related adverse outcomes in workers with anxiety disorders. Methods A systematic study search was conducted in Medline, Cinahl, Embase and Psycinfo. Two reviewers independently extracted data and from each study assessed the quality of evidence by using the GRADE approach. We performed a meta-analysis if data showed sufficient clinical homogeneity. Results Seven studies containing 11 exposure-in-vivo interventions were included. Four studies were focused on Obsessive Compulsive Disorder (OCD), two on Post Traumatic Stress Disorder (PTSD), and one on a mixed group of OCD and severe phobias. The studies were grouped according to type of anxiety disorder and subsequently according to type of comparisons. For OCD, exposure-in-vivo containing interventions can yield better work-related outcomes compared to medication (SSRIs) and relaxation but not better compared to response prevention. The results on anxiety outcomes were similar. The net contribution of exposure in vivo in two OCD intervention programs is also presented as a meta-analysis and shows significant positive results on work role limitations. The calculated pooled effect size with 95% confidence interval was 0.72 (0.28, 1.15). For PTSD, exposure-in-vivo containing interventions can yield better work-related and anxiety-related outcomes compared to a waiting-list but not better compared to imaginal exposure. Conclusions Exposure in vivo as part of an anxiety treatment can reduce work-related adverse outcomes in workers with OCD and PTSD better than various other anxiety treatments or a waiting-list. We recommend that it should be studied how the results of these studies can be transferred to the practice of occupational health professionals and how clinicians can make better use of them to improve work-related outcomes. In future research, priority should be given to high-quality randomised controlled trials (RCTs) in which exposure-in-vivo containing interventions are applied to a variety of anxiety disorders and compared with other clinical anxiety treatments such as SSRIs. Work-related outcomes, in particular work functioning and sickness absence, need to be assessed with reliable and valid measures.

2010-01-01

88

In vivo Analysis of Choroid Plexus Morphogenesis in Zebrafish  

PubMed Central

Background The choroid plexus (ChP), a component of the blood-brain barrier (BBB), produces the cerebrospinal fluid (CSF) and as a result plays a role in (i) protecting and nurturing the brain as well as (ii) in coordinating neuronal migration during neurodevelopment. Until now ChP development was not analyzed in living vertebrates due to technical problems. Methodology/Principal Findings We have analyzed the formation of the fourth ventricle ChP of zebrafish in the GFP-tagged enhancer trap transgenic line SqET33-E20 (Gateways) by a combination of in vivo imaging, histology and mutant analysis. This process includes the formation of the tela choroidea (TC), the recruitment of cells from rhombic lips and, finally, the coalescence of TC resulting in formation of ChP. In Notch-deficient mib mutants the first phase of this process is affected with premature GFP expression, deficient cell recruitment into TC and abnormal patterning of ChP. In Hedgehog-deficient smu mutants the second phase of the ChP morphogenesis lacks cell recruitment and TC cells undergo apoptosis. Conclusions/Significance This study is the first to demonstrate the formation of ChP in vivo revealing a role of Notch and Hedgehog signalling pathways during different developmental phases of this process.

Fong, Steven H.; Ye, Zhang-Rui; Korzh, Vladimir

2008-01-01

89

In vivo functional tests for assessing immunotoxicity in birds.  

PubMed

Various methods have been adapted for assessing the effects of environmental contaminants on the structure and function of the immune system in wild and captive birds. This chapter describes two integrative functional assays that have been adapted to a variety of avian species and have proven to be sensitive biomarkers for immunotoxicological effects. The phytohemagglutinin (PHA) skin test measures T cell-mediated immunity. PHA is injected intra- or sub-dermally into the wing web of the elbow joint (or interdigitary skin or wattle). The PHA stimulates T lymphocytes to release cytokines that cause an inflammatory influx of leukocytes and fluid. The thickness of the wing web is measured before and 24 h after injection. A stimulation index, which reflects T cell function, is calculated as the increase in skin thickness caused by the PHA minus the increase caused by an injection of phosphate buffered saline (PBS) in the other wing web. In addition to its sensitivity to contaminants, ecological studies have shown that the PHA skin response is positively associated with rates of survival and colonization of new areas (i.e., ability to found new local populations) in wild birds.The sheep red blood cell (SRBC) hemagglutination assay measures the antibody response to immunization with SRBC antigens, integrating the functions of B lymphocytes, helper T lymphocytes, and macrophages. A SRBC suspension is injected i.v., and a blood sample is collected approximately 6 days later. Plasma (or serum) from the blood sample is serially diluted in a microtiter plate, and SRBCs are added. The magnitude of the antibody response is defined as the titer - the highest dilution of plasma in which the concentration of antibody is sufficient to agglutinate the SRBCs. Both IgM and IgG titers can be measured. This avian test is very similar in principle to the anti-SRBC ELISA and splenic plaque forming assays used for immunotoxicological testing in rodents. However, this avian hemagglutination assay does not require a species-specific secondary antibody (as does the ELISA), and this minimally invasive, nonlethal procedure is amenable to studies of protected species, as opposed to the splenic assay. The PHA and SRBC assays have been employed successfully in both the laboratory and field. In ecological studies birds must be recaptured 24 h or 6 days after the initial injections, limiting their use in some species. However, their sensitivity to a variety of contaminants and their ease of adaptability to a variety of species have made the PHA and SRBC tests some of the most commonly used assays for screening and monitoring immunotoxicity in birds. PMID:19967526

Grasman, Keith A

2010-01-01

90

Glycerol accelerates recovery of barrier function in vivo.  

PubMed

Two studies were performed to evaluate the influence of glycerol on the recovery of damaged stratum corneum barrier function. Measurements of transepidermal water loss and capacitance were conducted in a 3-day follow-up after tape stripping (study 1) and a 7-day follow-up after a barrier damage due to a repeated washing with sodium lauryl sulphate. In study 1 a faster barrier repair (transepidermal water loss) was monitored in glycerol-treated sites. Significant differences between glycerol open vs. untreated and glycerol occluded vs. untreated were observed at day 3. Stratum corneum hydration showed significantly higher values in the sites treated with glycerol+occlusion, compared with all other sites. In study 2 a faster barrier repair was seen in glycerol-treated sites, with significant differences against untreated and base-treated sites 7 days after the end of the treatment. Stratum corneum hydration showed highest values in the glycerol treated sites after 3 days of treatment. Glycerol creates a stimulus for barrier repair and improves the stratum corneum hydration; stratum corneum hydration is not strictly related to barrier homeostasis and can be optimized by different mechanisms and pathways. The observed effects were based on the modulation of barrier repair and were not biased by the humectant effect of glycerol. As the glycerol-induced recovery of barrier function and stratum corneum hydration were observed even 7 days after the end of treatment, glycerol can be regarded as a barrier stabilizing and moisturizing compound. PMID:10598752

Fluhr, J W; Gloor, M; Lehmann, L; Lazzerini, S; Distante, F; Berardesca, E

1999-11-01

91

Effect of Processing and Storage on RBC function in vivo  

PubMed Central

Red Blood Cell (RBC) transfusion is indicated to improve oxygen delivery to tissue, and for no other purpose. We have come to appreciate that donor RBCs are fundamentally altered during processing and storage, in a fashion that both impairs oxygen transport efficacy and introduces additional risk by perturbing both immune and coagulation systems. The protean biophysical and physiologic changes in RBC function arising from storage are termed the ‘storage lesion’; many have been understood for some time; for example, we know that the oxygen affinity of stored blood rises during the storage period1 and that intracellular allosteric regulators, notably 2,3-bisphosphoglyceric acid (DPG) and ATP, are depleted during storage. Our appreciation of other storage lesion features has emerged with improved understanding of coagulation, immune and vascular signaling systems. Herein we review key features of the ‘storage lesion’. Additionally, we call particular attention to the newly appreciated role of RBCs in regulating linkage between regional blood flow and regional O2 consumption by regulating the bioavailability of key vasoactive mediators in plasma, as well as discuss how processing and storage disturbs this key signaling function and impairs transfusion efficacy.

Doctor, Allan; Spinella, Phil

2012-01-01

92

In vivo imaging of molecular targets and their function in endocrinology  

PubMed Central

Imaging is one of the fastest growing fields of study. New technologies and multimodal approaches are increasing the application of imaging to determine molecular targets and functional processes in vivo. The identification of a specific target, transporter, or biological process using imaging has introduced major breakthroughs to the field of endocrinology primarily utilizing computed tomography, magnetic resonance imaging, ultrasonography, positron emission tomography, single-photon emission computed tomography, and optical imaging. This review provides a general background to the specific developments in imaging that pertains to in vivo function and target identification in endocrine-based diseases.

Burdette, Joanna E

2010-01-01

93

SAHA Enhances Synaptic Function and Plasticity In Vitro but Has Limited Brain Availability In Vivo and Does Not Impact Cognition  

PubMed Central

Suberoylanilide hydroxamic acid (SAHA) is an inhibitor of histone deacetylases (HDACs) used for the treatment of cutaneous T cell lymphoma (CTCL) and under consideration for other indications. In vivo studies suggest reducing HDAC function can enhance synaptic function and memory, raising the possibility that SAHA treatment could have neurological benefits. We first examined the impacts of SAHA on synaptic function in vitro using rat organotypic hippocampal brain slices. Following several days of SAHA treatment, basal excitatory but not inhibitory synaptic function was enhanced. Presynaptic release probability and intrinsic neuronal excitability were unaffected suggesting SAHA treatment selectively enhanced postsynaptic excitatory function. In addition, long-term potentiation (LTP) of excitatory synapses was augmented, while long-term depression (LTD) was impaired in SAHA treated slices. Despite the in vitro synaptic enhancements, in vivo SAHA treatment did not rescue memory deficits in the Tg2576 mouse model of Alzheimer’s disease (AD). Along with the lack of behavioral impact, pharmacokinetic analysis indicated poor brain availability of SAHA. Broader assessment of in vivo SAHA treatment using high-content phenotypic characterization of C57Bl6 mice failed to demonstrate significant behavioral effects of up to 150 mg/kg SAHA following either acute or chronic injections. Potentially explaining the low brain exposure and lack of behavioral impacts, SAHA was found to be a substrate of the blood brain barrier (BBB) efflux transporters Pgp and Bcrp1. Thus while our in vitro data show that HDAC inhibition can enhance excitatory synaptic strength and potentiation, our in vivo data suggests limited brain availability may contribute to the lack of behavioral impact of SAHA following peripheral delivery. These results do not predict CNS effects of SAHA during clinical use and also emphasize the importance of analyzing brain drug levels when interpreting preclinical behavioral pharmacology.

Hanson, Jesse E.; La, Hank; Plise, Emile; Chen, Yung-Hsiang; Ding, Xiao; Hanania, Taleen; Sabath, Emily V.; Alexandrov, Vadim; Brunner, Dani; Leahy, Emer; Steiner, Pascal; Liu, Lichuan; Scearce-Levie, Kimberly; Zhou, Qiang

2013-01-01

94

Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions  

Microsoft Academic Search

Abp1 is a multidomain protein that regulates the Arp2\\/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here,

Omar Quintero-Monzon; Avital A. Rodal; Boris Strokopytov; Steven C. Almo; Bruce L. Goode

2005-01-01

95

Staged decline of neuronal function in vivo in an animal model of Alzheimer's disease  

PubMed Central

The accumulation of amyloid-? in the brain is an essential feature of Alzheimer's disease. However, the impact of amyloid-?-accumulation on neuronal dysfunction on the single cell level in vivo is poorly understood. Here we investigate the progression of amyloid-? load in relation to neuronal dysfunction in the visual system of the APP23×PS45 mouse model of Alzheimer's disease. Using in vivo two-photon calcium imaging in the visual cortex, we demonstrate that a progressive deterioration of neuronal tuning for the orientation of visual stimuli occurs in parallel with the age-dependent increase of the amyloid-? load. Importantly, we find this deterioration only in neurons that are hyperactive during spontaneous activity. This impairment of visual cortical circuit function also correlates with pronounced deficits in visual-pattern discrimination. Together, our results identify distinct stages of decline in sensory cortical performance in vivo as a function of the increased amyloid-?-load.

Grienberger, Christine; Rochefort, Nathalie L.; Adelsberger, Helmuth; Henning, Horst A.; Hill, Daniel N.; Reichwald, Julia; Staufenbiel, Matthias; Konnerth, Arthur

2012-01-01

96

In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.  

PubMed

The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs ((3)H-HPG) of different molecular weights; the values ranged from 1 × 10(5) to 2 × 10(6) molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 10(5) HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of (3)H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of (3)H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of (3)H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC. PMID:22261097

Chapanian, Rafi; Constantinescu, Iren; Brooks, Donald E; Scott, Mark D; Kizhakkedathu, Jayachandran N

2012-01-18

97

EVENT PLANNING USING FUNCTION ANALYSIS  

Microsoft Academic Search

Event planning is expensive and resource intensive. Function analysis provides a solid foundation for comprehensive event planning (e.g., workshops, conferences, symposiums, or meetings). It has been used at Idaho National Laboratory (INL) to successfully plan events and capture lessons learned, and played a significant role in the development and implementation of the âINL Guide for Hosting an Event.âť Using a

Lori Braase; Jodi Grgich

2011-01-01

98

A chemical-genetic approach to study G protein regulation of ? cell function in vivo  

PubMed Central

Impaired functioning of pancreatic ? cells is a key hallmark of type 2 diabetes. ? cell function is modulated by the actions of different classes of heterotrimeric G proteins. The functional consequences of activating specific ? cell G protein signaling pathways in vivo are not well understood at present, primarily due to the fact that ? cell G protein-coupled receptors (GPCRs) are also expressed by many other tissues. To circumvent these difficulties, we developed a chemical-genetic approach that allows for the conditional and selective activation of specific ? cell G proteins in intact animals. Specifically, we created two lines of transgenic mice each of which expressed a specific designer GPCR in ? cells only. Importantly, the two designer receptors differed in their G protein-coupling properties (Gq/11 versus Gs). They were unable to bind endogenous ligand(s), but could be efficiently activated by an otherwise pharmacologically inert compound (clozapine-N-oxide), leading to the conditional activation of either ? cell Gq/11 or Gs G proteins. Here we report the findings that conditional and selective activation of ? cell Gq/11 signaling in vivo leads to striking increases in both first- and second-phase insulin release, greatly improved glucose tolerance in obese, insulin-resistant mice, and elevated ? cell mass, associated with pathway-specific alterations in islet gene expression levels. Selective stimulation of ? cell Gs triggered qualitatively similar in vivo metabolic effects. Thus, this developed chemical-genetic strategy represents a powerful approach to study G protein regulation of ? cell function in vivo.

Guettier, Jean-Marc; Gautam, Dinesh; Scarselli, Marco; de Azua, Inigo Ruiz; Li, Jian Hua; Rosemond, Erica; Ma, Xiaochao; Gonzalez, Frank J.; Armbruster, Blaine N.; Lu, Huiyan; Roth, Bryan L.; Wess, Jurgen

2009-01-01

99

In vivo imaging of neutrotransmitter functions in brain, heart and tumors. Proceedings  

SciTech Connect

This volume contains the proceedings of a symposium entitled ``In Vivo Imaging of Neurotransmitter Function in Brain, Heart, and Tumors`` held August 24--25, 1990 in Montreal Canada. The six individual papers contained herein are separately abstracted and indexed for the database.

Kuhl, D.E. [ed.

1991-12-31

100

In vivo imaging of neutrotransmitter functions in brain, heart and tumors  

SciTech Connect

This volume contains the proceedings of a symposium entitled In Vivo Imaging of Neurotransmitter Function in Brain, Heart, and Tumors'' held August 24--25, 1990 in Montreal Canada. The six individual papers contained herein are separately abstracted and indexed for the database.

Kuhl, D.E. (ed.)

1991-01-01

101

A Sensitive in vivo Platelet Function Test in Rats Based on Intravenously Injected Collagenase  

Microsoft Academic Search

The transient decrease of the platelet concentration in the flowing blood after intravenous injection of collagenase was used as a measure for the effectiveness of the platelet-vessel wall interactions in rats. The decrease of the concentration in circulating platelets was regarded as an expression of the in vivo platelet function. The influence of acetylsalicylic acid, imidazole, indomethacin, ketanserin, RA 233,

K.-P. Völkl

1989-01-01

102

Ageing-related changes in the in vivo function of rat liver macroautophagy and proteolysis  

Microsoft Academic Search

Autophagy is a universal, highly regulated mechanism responsible for the degradation of long-lived proteins, cytomembranes and organelles during fasting and may be the cell repair mechanism that mediates the anti-ageing effects of calorie restriction (Bergamini and Gori, 1995). The function of autophagy was studied in vivo on male Sprague Dawley rats fed ad libitum or 40% food restricted. Autophagy was

Alessandra Del Roso; Simona Vittorini; Gabriella Cavallini; Alessio Donati; Zina Gori; Matilde Masini; Maria Pollera; Ettore Bergamini

2003-01-01

103

EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT DURING PREGNANCY  

EPA Science Inventory

Effects of Bromodichloromethane (BDCM) on Ex Vivo Luteal Function In the Pregnant F344 Rat Susan R. Bielmeier1, Ashley S. Murr2, Deborah S. Best2, Jerome M. Goldman2, and Michael G. Narotsky2 1Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC 27599,...

104

Experimental models to study development and function of the human immune system in vivo  

Microsoft Academic Search

The study of development and function of the immune system in vivo has made intensive use ofanimal models, but performing such work in humans is difflcultfor experimental, practical and ethical reasons. Conftonted with this scientific challenge, several pioneering groups have developed in the late 1980s mouse models ofhuman immune system development. Although these experimental approaches were proven successful and useful

Nicolas Legrand; Kees Weijer; Hergen Spits

2006-01-01

105

Recent developments in the understanding of astrocyte function in the cerebellum in vivo.  

PubMed

Several studies have contributed to our understanding of astrocytes, especially Bergmann glia, in the cerebellum; but, until recently, none has looked at their function in vivo. Multicell bolus loading of fluorescent calcium indicators in combination with the astrocytic marker SR101 has allowed imaging of up to hundreds of astrocytes at once in the intact cerebellum. In addition, the selective targeting of astrocytes with fluorescent calcium indicator proteins has enabled the study of their function in vivo without the confounding effects of other neuropil signals and with a resolution that surpasses multicell bolus loading and SR101 staining. The two astrocyte types of the cerebellar cortex, Bergmann glia, and velate protoplasmic astrocytes display a diverse signaling repertoire in vivo, which ranges from localized calcium elevations in subcellular processes to waves, triggered by the release of purines and mediated by purinergic receptors that span multiple processes and can involve tens of astrocytes. During locomotor behavior, even larger numbers of astrocytes display calcium increases that are driven by neuronal activity and correlate with global changes in blood flow. In this review, we give an overview of our current understanding of the function of Bergmann glia and velate protoplasmic astrocytes and the promise of the tools used to study their calcium dynamics and function in vivo. PMID:19904577

Hoogland, Tycho M; Kuhn, Bernd

2010-09-01

106

Quantifying long-term microelectrode array functionality using chronic in vivo impedance testing  

NASA Astrophysics Data System (ADS)

Long-term acquisition of high-quality neural recordings is a cornerstone of neuroprosthetic system design. Mitigating the experimental variability of chronically implanted arrays has been a formidable task because the sensor recording sites can be influenced by biotic and abiotic responses. Several studies have implicated changes in electrical interface impedance as a preliminary marker to infer electrode viability. Microelectrode impedance plays an important role in the monitoring of low amplitude and high-resolution extracellular neural signals. In this work, we seek to quantify long-term microelectrode array functionality and derive an impedance-based predictor for electrode functionality that correlates the recording site electrical properties with the functional neuronal recordings in vivo. High temporal resolution metrics of this type would allow one to assess, predict, and improve electrode performance in the future. In a large cohort of animals, we performed daily impedance measurements and neural signal recordings over long periods (up to 21 weeks) of time in rats using tungsten microwire arrays implanted into the somatosensory cortex. This study revealed that there was a time-varying trend in the modulation of impedance that was related to electrode performance. Single units were best detected from electrodes at time points when the electrode entered into the 40-150 K? impedance range. This impedance trend was modeled across the full cohort of animals to predict future electrode performance. The model was tested on data from all animals and was able to provide predictions of electrode performance chronically. Insight from this study can be combined with knowledge of electrode materials and histological analysis to provide a more comprehensive predictive model of electrode failure in the future.

Prasad, Abhishek; Sanchez, Justin C.

2012-04-01

107

Quantifying long-term microelectrode array functionality using chronic in vivo impedance testing.  

PubMed

Long-term acquisition of high-quality neural recordings is a cornerstone of neuroprosthetic system design. Mitigating the experimental variability of chronically implanted arrays has been a formidable task because the sensor recording sites can be influenced by biotic and abiotic responses. Several studies have implicated changes in electrical interface impedance as a preliminary marker to infer electrode viability. Microelectrode impedance plays an important role in the monitoring of low amplitude and high-resolution extracellular neural signals. In this work, we seek to quantify long-term microelectrode array functionality and derive an impedance-based predictor for electrode functionality that correlates the recording site electrical properties with the functional neuronal recordings in vivo. High temporal resolution metrics of this type would allow one to assess, predict, and improve electrode performance in the future. In a large cohort of animals, we performed daily impedance measurements and neural signal recordings over long periods (up to 21 weeks) of time in rats using tungsten microwire arrays implanted into the somatosensory cortex. This study revealed that there was a time-varying trend in the modulation of impedance that was related to electrode performance. Single units were best detected from electrodes at time points when the electrode entered into the 40-150 K? impedance range. This impedance trend was modeled across the full cohort of animals to predict future electrode performance. The model was tested on data from all animals and was able to provide predictions of electrode performance chronically. Insight from this study can be combined with knowledge of electrode materials and histological analysis to provide a more comprehensive predictive model of electrode failure in the future. PMID:22442134

Prasad, Abhishek; Sanchez, Justin C

2012-03-23

108

Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo  

PubMed Central

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild type E. coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and 15N-labeled wild type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse labeling with 15N-labeled medium timestamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation, or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.

Sykes, Michael T.; Shajani, Zahra; Sperling, Edit; Beck, Andrea H.; Williamson, James R.

2010-01-01

109

Functional Multiple-Set Canonical Correlation Analysis  

ERIC Educational Resources Information Center

|We propose functional multiple-set canonical correlation analysis for exploring associations among multiple sets of functions. The proposed method includes functional canonical correlation analysis as a special case when only two sets of functions are considered. As in classical multiple-set canonical correlation analysis, computationally, the…

Hwang, Heungsun; Jung, Kwanghee; Takane, Yoshio; Woodward, Todd S.

2012-01-01

110

Functional Multiple-Set Canonical Correlation Analysis  

ERIC Educational Resources Information Center

We propose functional multiple-set canonical correlation analysis for exploring associations among multiple sets of functions. The proposed method includes functional canonical correlation analysis as a special case when only two sets of functions are considered. As in classical multiple-set canonical correlation analysis, computationally, the…

Hwang, Heungsun; Jung, Kwanghee; Takane, Yoshio; Woodward, Todd S.

2012-01-01

111

The proximal element of the beta globin locus control region is not functionally required in vivo.  

PubMed Central

In addition to local sequence elements the regulation of the high-level, development- and tissue-specific expression of the human beta globin gene cluster appears to require distant regulatory sequences which have been termed locus control region. In the chromatin of erythroid cells the locus control region is characterized by four DNaseI hypersensitive sites that are located 6-18 kb 5' of the epsilon globin gene. The definition of the sequences minimally required for locus control region activity is likely to further the understanding of its physiology and will be of interest for the development of somatic gene therapy strategies of the hemoglobinopathies. We present here the analysis of a family with a 3,030-bp deletion of sequences upstream of the epsilon globin gene including the most 3' locus control region element and cosegregating beta(0) thalassemia. The deletion is linked in cis to a structurally and functionally normal beta globin gene. The proximal element of the locus control region does not therefore appear to be necessary for beta globin gene activity in vivo. Images

Kulozik, A E; Bail, S; Bellan-Koch, A; Bartram, C R; Kohne, E; Kleihauer, E

1991-01-01

112

B Plant function analysis report  

SciTech Connect

The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and a function hierarchy chart that describe what needs to be performed to deactivate B Plant.

Lund, D.P.; B Plant Working Group

1995-09-01

113

Transcriptional Regulation of Rod Photoreceptor Homeostasis Revealed by In Vivo NRL Targetome Analysis  

PubMed Central

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP–Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP–Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP–Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.

Hao, Hong; Kim, Douglas S.; Klocke, Bernward; Johnson, Kory R.; Cui, Kairong; Gotoh, Norimoto; Zang, Chongzhi; Gregorski, Janina; Gieser, Linn; Peng, Weiqun; Fann, Yang; Seifert, Martin; Zhao, Keji; Swaroop, Anand

2012-01-01

114

In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8[W  

PubMed Central

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the ?-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function.

O'Hara, Andrew; Jenkins, Gareth I.

2012-01-01

115

Optogenetic analysis of synaptic function.  

PubMed

We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or gamma-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for gamma-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evoked neurotransmitter-specific postsynaptic currents that could be triggered repeatedly and at various frequencies. Light-evoked behaviors and postsynaptic currents were significantly (P function in all cases tested. OptIoN facilitates the analysis of neurotransmission with high temporal precision, in a neurotransmitter-selective manner, possibly allowing future investigation of synaptic plasticity in C. elegans. PMID:18794862

Liewald, Jana F; Brauner, Martin; Stephens, Greg J; Bouhours, Magali; Schultheis, Christian; Zhen, Mei; Gottschalk, Alexander

2008-09-14

116

Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.  

PubMed Central

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated. Images

Hutchens, J A; Hoyle, H D; Turner, F R; Raff, E C

1997-01-01

117

Functionalized gold nanoparticles: a detailed in vivo multimodal microscopic brain distribution study  

NASA Astrophysics Data System (ADS)

In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex.In the present study, the in vivo distribution of polyelectrolyte multilayer coated gold nanoparticles is shown, starting from the living animal down to cellular level. The coating was designed with functional moieties to serve as a potential nano drug for prion disease. With near infrared time-domain imaging we followed the biodistribution in mice up to 7 days after intravenous injection of the nanoparticles. The peak concentration in the head of mice was detected between 19 and 24 h. The precise particle distribution in the brain was studied ex vivo by X-ray microtomography, confocal laser and fluorescence microscopy. We found that the particles mainly accumulate in the hippocampus, thalamus, hypothalamus, and the cerebral cortex. Electronic supplementary information (ESI) available: Fig. S1-S6. See DOI: 10.1039/c0nr00345j

Sousa, Fernanda; Mandal, Subhra; Garrovo, Chiara; Astolfo, Alberto; Bonifacio, Alois; Latawiec, Diane; Menk, Ralf Hendrik; Arfelli, Fulvia; Huewel, Sabine; Legname, Giuseppe; Galla, Hans-Joachim; Krol, Silke

2010-12-01

118

Effect of in vivo chronic exposure to clotrimazole on zebrafish testis function.  

PubMed

Clotrimazole is an azole fungicide used as a human pharmaceutical that is known to inhibit cytochrome P450 (CYP) enzymatic activities, including several steroidogenic CYP. In a previous report, we showed that a 7-day exposure to clotrimazole induced the expression of genes related to steroidogenesis in the testes as a compensatory response, involving the activation of the Fsh/Fshr pathway. In this context, the aim of the present study was to assess the effect of an in vivo 21-day chronic exposure to clotrimazole (30-197 ?g/L) on zebrafish testis function, i.e., spermatogenesis and androgen release. The experimental design combined (1) gene transcript levels measurements along the brain-pituitary-gonad axis, (2) 11-ketotestosterone (11-KT) quantification in the blood, and (3) histology of the testes, including morphometric analysis. The chronic exposure led to an induction of steroidogenesis-related genes and fshr in the testes as well as fsh? in the pituitary. Moreover, increases of the gonadosomatic index and of the volume proportion of interstitial Leydig cells were observed in clotrimazole-exposed fish. In accordance with these histological observations, the circulating concentration of 11-KT had increased. Morphometric analysis of the testes did not show an effect of clotrimazole on meiotic (spermatocytes) or postmeiotic (spermatids and spermatozoa) stages, but we observed an increase in the number of type A spermatogonia, in agreement with an increase in mRNA levels of piwil1, a specific molecular marker of type A spermatogonia. Our study demonstrated that clotrimazole is able to affect testicular physiology and raised further concern about the impact of clotrimazole on reproduction. PMID:23340899

Baudiffier, Damien; Hinfray, Nathalie; Ravaud, Catherine; Creusot, Nicolas; Chadili, Edith; Porcher, Jean-Marc; Schulz, Rüdiger W; Brion, François

2013-01-23

119

In vivo kinematic analysis of squatting after total hip arthroplasty  

Microsoft Academic Search

BackgroundThe in vivo kinematics of squatting after total hip arthroplasty is unclear. The purpose of the present study was to determine the range of motion of the hip joint during squatting after total hip arthroplasty.

Junichiro Koyanagi; Takashi Sakai; Takaharu Yamazaki; Tetsu Watanabe; Keisuke Akiyama; Nobuhiko Sugano; Hideki Yoshikawa; Kazuomi Sugamoto

2011-01-01

120

Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity.  

PubMed

Thiamin (vitamin B1) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms' environment and the particular pathway used. PMID:23959877

Moulin, Michael; Nguyen, Ginnie T D T; Scaife, Mark A; Smith, Alison G; Fitzpatrick, Teresa B

2013-08-19

121

Skeletal muscle oxidative function in vivo and ex vivo in athletes with marked hypertrophy from resistance training.  

PubMed

Oxidative function during exercise was evaluated in 11 young athletes with marked skeletal muscle hypertrophy induced by long-term resistance training (RTA; body mass 102.6 ± 7.3 kg, mean ± SD) and 11 controls (CTRL; body mass 77.8 ± 6.0 kg). Pulmonary O2 uptake (Vo2) and vastus lateralis muscle fractional O2 extraction (by near-infrared spectroscopy) were determined during an incremental cycle ergometer (CE) and one-leg knee-extension (KE) exercise. Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers obtained by biopsy. Quadriceps femoris muscle cross-sectional area, volume (determined by magnetic resonance imaging), and strength were greater in RTA vs. CTRL (by ?40%, ?33%, and ?20%, respectively). Vo2peak during CE was higher in RTA vs. CTRL (4.05 ± 0.64 vs. 3.56 ± 0.30 l/min); no difference between groups was observed during KE. The O2 cost of CE exercise was not different between groups. When divided per muscle mass (for CE) or quadriceps muscle mass (for KE), Vo2 peak was lower (by 15-20%) in RTA vs. CTRL. Vastus lateralis fractional O2 extraction was lower in RTA vs. CTRL at all work rates, during both CE and KE. RTA had higher ADP-stimulated mitochondrial respiration (56.7 ± 23.7 pmol O2·s(-1)·mg(-1) ww) vs. CTRL (35.7 ± 10.2 pmol O2·s(-1)·mg(-1) ww) and a tighter coupling of oxidative phosphorylation. In RTA, the greater muscle mass and maximal force and the enhanced mitochondrial respiration seem to compensate for the hypertrophy-induced impaired peripheral O2 diffusion. The net results are an enhanced whole body oxidative function at peak exercise and unchanged efficiency and O2 cost at submaximal exercise, despite a much greater body mass. PMID:23519233

Salvadego, Desy; Domenis, Rossana; Lazzer, Stefano; Porcelli, Simone; Rittweger, Jörn; Rizzo, Giovanna; Mavelli, Irene; Simunic, Bostjan; Pisot, Rado; Grassi, Bruno

2013-03-21

122

Functional Characterization of Antibodies Neutralizing Soluble Factors In Vitro and In Vivo  

Microsoft Academic Search

\\u000a Functional characterization of antibodies that inhibit soluble cytokines or chemokines requires robust, sensitive in vitro\\u000a and in vivo bioassays. Testing an antibody in vitro requires consideration of antigen source, integrity, and concentration,\\u000a as well as the magnitude of the biologic response and assay interference by components in the antibody test sample. This chapter\\u000a describes several exemplary in vitro bioassays, including

Geertruida M. Veldman; Zehra Kaymakcalan; Renee Miller; Leena Kalghatgi; Jochen G. Salfeld

123

In vivo migration and function of transferred HIV1-specific cytotoxic T cells  

Microsoft Academic Search

The persistence of HIV replication in infected individuals may reflect an inadequate host HIV-specific CD8+ cytotoxic T lymphocyte (CTL) response. The functional activity of HIV-specific CTLs and the ability of these effector cells to migrate in vivo to sites of infection was directly assessed by expanding autologous HIV-1 Gag-specific CD8+ CTL clones in vitro and adoptively transferring these CTLs to

Scott J. Brodie; Deborah A. Lewinsohn; Bruce K. Patterson; Daniel Jiyamapa; John Krieger; Lawrence Corey; Philip D. Greenberg; Stanley R. Riddell

1999-01-01

124

In Vivo Functional Assay of a Recombinant Aquaporin in Pichia pastoris  

Microsoft Academic Search

The water channel protein PvTIP3;1 (-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay

Mark J. Daniels; Malcolm R. Wood; Mark Yeager

2006-01-01

125

In vivo studies of development of the main functional systems in the heteronemertean pilidium larva  

Microsoft Academic Search

There is performed in vivo morphological study of the White Sea heteronemertines belonging to the type of Pilidium pyramidale (conussoidale). Based on the layer-by-layer microshooting with subsequent computer processing, development of the pilidium digestive, nervous,\\u000a and muscle systems is described from the stage following at once the gastrula till the premetamorphosis larva. Peculiarities\\u000a of structural organization of the main functional

O. V. Zaitseva; L. P. Flyachinskaya

2010-01-01

126

Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo  

Microsoft Academic Search

Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in

Richard Benton; Silke Sachse; Stephen W. Michnick; Leslie B. Vosshall

2006-01-01

127

Recent Developments in the Understanding of Astrocyte Function in the Cerebellum In Vivo  

Microsoft Academic Search

Several studies have contributed to our understanding of astrocytes, especially Bergmann glia, in the cerebellum; but, until\\u000a recently, none has looked at their function in vivo. Multicell bolus loading of fluorescent calcium indicators in combination\\u000a with the astrocytic marker SR101 has allowed imaging of up to hundreds of astrocytes at once in the intact cerebellum. In\\u000a addition, the selective targeting

Tycho M. Hoogland; Bernd Kuhn

2010-01-01

128

In vivo Testing of Functional Properties of Three Selected Probiotic Strains  

Microsoft Academic Search

Summary  Lactobacillus acidophilus M92, Lactobacillus plantarum L4 and Enterococcus faecium L3 were previously selected as probiotic strains on the base of in vitro selection criteria. To investigate functional properties of these three probiotic strains in vivo, Swiss albino mice were used as animal model. Survival, competition, adhesion and colonization were monitored in the gastrointestinal\\u000a tract, as well as the immunomodulating capability

J. Frece; B. Kos; J. Beganovi?; S. Vukovi?; J. Šuškovi?

2005-01-01

129

Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions  

SciTech Connect

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

2005-01-01

130

Functional Analysis and Treatment of Nail Biting  

ERIC Educational Resources Information Center

This study applied functional analysis methodology to nail biting exhibited by a 24-year-old female graduate student. Results from the brief functional analysis indicated variability in nail biting across assessment conditions. Functional analysis data were then used to guide treatment development and implementation. Treatment included a…

Dufrene, Brad A.; Watson, T. Steuart; Kazmerski, Jennifer S.

2008-01-01

131

On properties of functional principal components analysis  

Microsoft Academic Search

Functional data analysis is intrinsically infinite dimensional; functional principal component analysis reduces dimension to a finite level, and points to the most significant components of the data. However, although this technique is often discussed, its properties are not as well understood as they might be. We show how the properties of functional principal component analysis can be elucidated through stochastic

Peter Hall; Mohammad Hosseini-Nasab

2006-01-01

132

Some current themes in functional analysis research  

Microsoft Academic Search

The purpose of this article is to review and discuss some current themes in functional analysis research. The paper is divided into two general sections; one section discusses clinical application of functional analysis and a second section discusses functional analysis as a research method. In the first section, current issues related to treatment logic and development are reviewed. Also, clinical

Richard G. Smith

1996-01-01

133

Non-invasive in vivo imaging of pancreatic ?-cell function and survival - a perspective.  

PubMed

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/?-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic ?-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional ?-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus. PMID:21477063

Leibiger, I B; Caicedo, A; Berggren, P-O

2011-05-28

134

Non-invasive in vivo imaging of pancreatic ?-cell function and survival - a perspective  

PubMed Central

A major problem in medical research is to translate in vitro observations into the living organism. In this perspective, we discuss ongoing efforts to non-invasively image pancreatic islets/?-cells by techniques, such as magnetic resonance imaging and positron emission tomography, and present an experimental platform, which allows in vivo imaging of pancreatic ?-cell mass and function longitudinally and at the single-cell level. Following transplantation of pancreatic islets into the anterior chamber of the eye of mice and rats, these islets are studied by functional microscopic imaging. This imaging platform can be utilized to address fundamental aspects of pancreatic islet cell biology in vivo in health and disease. These include the dynamics of pancreatic islet vascularization, islet cell innervation, signal-transduction, change in functional ?-cell mass and immune responses. Moreover, we discuss the feasibility of studying human islet cell physiology and pathology in vivo as well as the potential of using the anterior chamber of the eye as a site for therapeutic transplantation in type 1 diabetes mellitus.

Leibiger, I. B.; Caicedo, A.; Berggren, P.-O.

2012-01-01

135

Direct link between RACK1 function and localization at the ribosome in vivo.  

PubMed

The receptor for activated C-kinase (RACK1), a conserved protein implicated in numerous signaling pathways, is a stoichiometric component of eukaryotic ribosomes located on the head of the 40S ribosomal subunit. To test the hypothesis that ribosome association is central to the function of RACK1 in vivo, we determined the 2.1-A crystal structure of RACK1 from Saccharomyces cerevisiae (Asc1p) and used it to design eight mutant versions of RACK1 to assess roles in ribosome binding and in vivo function. Conserved charged amino acids on one side of the beta-propeller structure were found to confer most of the 40S subunit binding affinity, whereas an adjacent conserved and structured loop had little effect on RACK1-ribosome association. Yeast mutations that confer moderate to strong defects in ribosome binding mimic some phenotypes of a RACK1 deletion strain, including increased sensitivity to drugs affecting cell wall biosynthesis and translation elongation. Furthermore, disruption of RACK1's position at the 40S ribosomal subunit results in the failure of the mRNA binding protein Scp160 to associate with actively translating ribosomes. These results provide the first direct evidence that RACK1 functions from the ribosome, implying a physical link between the eukaryotic ribosome and cell signaling pathways in vivo. PMID:19114558

Coyle, Scott M; Gilbert, Wendy V; Doudna, Jennifer A

2008-12-29

136

In Vivo Neutron Activation Analysis: Body Composition Studies in Health and Disease.  

National Technical Information Service (NTIS)

In vivo analysis of body elements by neutron activation is an important tool in medical research. It has provided a direct quantitative measure of body composition of human beings in vivo. Basic physiological differences related to age, sex, race, and bod...

K. J. Ellis S. H. Cohn

1984-01-01

137

Nucleotide binding by Lhs1p is essential for its nucleotide exchange activity and for function in vivo.  

PubMed

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo. PMID:19759005

de Keyzer, Jeanine; Steel, Gregor J; Hale, Sarah J; Humphries, Daniel; Stirling, Colin J

2009-09-15

138

Maternal separation affects dopamine transporter function in the Spontaneously Hypertensive Rat: An in vivo electrochemical study  

PubMed Central

Background Attention-deficit/hyperactivity disorder (ADHD) is a developmental disorder characterised by symptoms of inattention, impulsivity and hyperactivity. The spontaneously hypertensive rat (SHR) is a well-characterised model of this disorder and has been shown to exhibit dopamine dysregulation, one of the hypothesised causes of ADHD. Since stress experienced in the early stages of life can have long-lasting effects on behaviour, it was considered that early life stress may alter development of the dopaminergic system and thereby contribute to the behavioural characteristics of SHR. It was hypothesized that maternal separation would alter dopamine regulation by the transporter (DAT) in ways that distinguish SHR from control rat strains. Methods SHR and control Wistar-Kyoto (WKY) rats were subjected to maternal separation for 3 hours per day from postnatal day 2 to 14. Rats were tested for separation-induced anxiety-like behaviour followed by in vivo chronoamperometry to determine whether changes had occurred in striatal clearance of dopamine by DAT. The rate of disappearance of ejected dopamine was used as a measure of DAT function. Results Consistent with a model for ADHD, SHR were more active than WKY in the open field. SHR entered the inner zone more frequently and covered a significantly greater distance than WKY. Maternal separation increased the time that WKY spent in the closed arms and latency to enter the open arms of the elevated plus maze, consistent with other rat strains. Of note is that, maternal separation failed to produce anxiety-like behaviour in SHR. Analysis of the chronoamperometric data revealed that there was no difference in DAT function in the striatum of non-separated SHR and WKY. Maternal separation decreased the rate of dopamine clearance (k-1) in SHR striatum. Consistent with this observation, the dopamine clearance time (T100) was increased in SHR. These results suggest that the chronic mild stress of maternal separation impaired the function of striatal DAT in SHR. Conclusions The present findings suggest that maternal separation failed to alter the behaviour of SHR in the open field and elevated plus maze. However, maternal separation altered the dopaminergic system by decreasing surface expression of DAT and/or the affinity of DAT for dopamine, increasing the time to clear dopamine from the extracellular fluid in the striatum of SHR.

2011-01-01

139

Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo  

PubMed Central

Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown Norway rats were dosed (8 mg/kg, i.t.) 24 hr prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed.

Lord, Kevin; Moll, David; Lindsey, John K.; Mahne, Sarah; Raman, Girija; Dugas, Tammy; Cormier, Stephania; Troxlair, Dana; Lomnicki, Slawo; Dellinger, Barry; Varner, Kurt

2011-01-01

140

Environmentally persistent free radicals decrease cardiac function before and after ischemia/reperfusion injury in vivo.  

PubMed

Exposure to airborne particles is associated with increased cardiovascular morbidity and mortality. During the combustion of chlorine-containing hazardous materials and fuels, chlorinated hydrocarbons chemisorb to the surface of transition metal-oxide-containing particles, reduce the metal, and form an organic free radical. These radical-particle systems can survive in the environment for days and are called environmentally persistent free radicals (EPFRs). This study determined whether EPFRs could decrease left ventricular function before and after ischemia and reperfusion (I/R) in vivo. Male Brown-Norway rats were dosed (8?mg/kg, intratracheal) 24?h prior to testing with particles containing the EPFR of 1, 2-dichlorobenzene (DCB230). DCB230 treatment decreased systolic and diastolic function. DCB230 also produced pulmonary and cardiac inflammation. After ischemia, systolic, but not diastolic function was significantly decreased in DCB230-treated rats. Ventricular function was not affected by I/R in control rats. There was greater oxidative stress in the heart and increased 8-isoprostane (biomarker of oxidative stress) in the plasma of treated vs. control rats after I/R. These data demonstrate for the first time that DCB230 can produce inflammation and significantly decrease cardiac function at baseline and after I/R in vivo. Furthermore, these data suggest that EPFRs may be a risk factor for cardiac toxicity in healthy individuals and individuals with ischemic heart disease. Potential mechanisms involving cytokines/chemokines and/or oxidative stress are discussed. PMID:21385100

Lord, Kevin; Moll, David; Lindsey, John K; Mahne, Sarah; Raman, Girija; Dugas, Tammy; Cormier, Stephania; Troxlair, Dana; Lomnicki, Slawo; Dellinger, Barry; Varner, Kurt

2011-04-01

141

Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo  

PubMed Central

The 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, in addition to providing a 16-nt template region for reverse transcription, has been proposed to act as a scaffold for protein subunits. Although accessory subunits of the telomerase ribonucleoprotein (RNP) complex function even when their binding sites are relocated on the yeast telomerase RNA, the physical nature of the RNA scaffold has not been directly analyzed. Here we explore the structure–function organization of the yeast telomerase RNP by extensively stiffening the three long arms of TLC1, which connect essential and important accessory protein subunits Ku, Est1, and Sm7, to its central catalytic hub. This 956-nt triple-stiff-arm TLC1 (TSA-T) reconstitutes active telomerase with TERT (Est2) in vitro. Furthermore, TSA-T functions in vivo, even maintaining longer telomeres than TLC1 on a per RNA basis. We also tested functional contributions of each stiffened arm within TSA-T and found that the stiffened Est1 and Ku arms contribute to telomere lengthening, while stiffening the terminal arm reduces telomere length and telomerase RNA abundance. The fact that yeast telomerase tolerates significant stiffening of its RNA subunit in vivo advances our understanding of the architectural and functional organization of this RNP and, more broadly, our conception of the world of lncRNPs.

Lebo, Kevin J.; Zappulla, David C.

2012-01-01

142

Harmonic Analysis of Polynomial Threshold Functions  

Microsoft Academic Search

The analysis of linear threshold Boolean functions has recently attracted the attention of those interested in circuit complexity as well as of those interested in neural networks. Here a generalization of linear threshold functions is defined, namely, polynomial threshold functions, and its relation to the class of linear threshold functions is investigated. A Boolean function is polynomial threshold if it

Jehoshua Bruck

1990-01-01

143

Hepatitis C Virus-Infected Cells Downregulate NKp30 and Inhibit Ex Vivo NK Cell Functions.  

PubMed

Hepatitis C virus (HCV) successfully evades the immune system and establishes chronic infection in ?80% of cases. Immune evasion may involve modulating NK cell functions. Therefore, we developed a short-term assay to assess immediate effects of HCV-infected cells on ex vivo NK cytotoxicity and cytokine production. Natural cytotoxicity, Ab-dependent cell-mediated cytotoxicity, IFN-? production, and TNF-? production were all significantly inhibited by short-term direct exposure to HCV-infected hepatoma-derived Huh-7.5 cells. Inhibition required cell-to-cell contact and increased together with multiplicity of infection and HCV protein levels. Blocking potential interaction between HCV E2 and NK CD81 did not abrogate NK cell inhibition mediated by HCV-infected cells. We observed no change in expression levels of NKG2D, NKG2A, NKp46, or CD16 on NK cells exposed to HCV-infected Huh-7.5 cells for 5 h or of human histocompatibility-linked leukocyte Ag E on HCV-infected compared with uninfected Huh-7.5 cells. Inhibition of ex vivo NK functions did correspond with reduced surface expression of the natural cytotoxicity receptor NKp30, and downregulation of NKp30 was functionally reflected in reduced anti-NKp30 redirected lysis of P815 cells. Infection of Huh-7.5 cells with HCV JFH1T increased surface binding of an NKp30-IgG1 Fc? fusion protein, suggesting upregulation of an antagonistic NKp30 ligand on HCV-infected cells. Our assay demonstrates rapid inhibition of critical NK cell functions by HCV-infected cells. Similar localized effects in vivo may contribute to establishment of chronic HCV infection and associated phenotypic and functional changes in the NK population. PMID:23960237

Holder, Kayla A; Stapleton, Staci N; Gallant, Maureen E; Russell, Rodney S; Grant, Michael D

2013-08-19

144

Longitudinal assessment of endothelial function in the microvasculature of mice in-vivo.  

PubMed

Endothelial dysfunction is associated with early development of cardiovascular disease, making longitudinal measurements desirable. We devised a protocol using laser Doppler imaging (LDI) and iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) to assess the skin microcirculation longitudinally in mice every 4 weeks for 24 weeks in two groups of C57BL/6 mice, chow versus high-cholesterol diet(known to induce endothelial dysfunction). LDI measurements were compared with vascular function (isometric tension) measured using wire myography in the tail artery in response to ACh and SNP. Microvascular responses to ACh were significantly reduced in cholesterol-fed versus chow-fed mice from week 4 onwards (P<0.005, ANOVA). Pre-treatment with N(G)-nitro-L-arginine methyl-ester-hydrochloride (L-NAME) showed a significant reduction in ACh response compared with vehicle-treated animals (P<0.05) at baseline and at 12 weeks. In cholesterol-fed mice, ACh responses were 226 ± 21 and 180 ± 21 AU (P=0.03) before and after L-NAME, respectively. A reduction in ex-vivo ACh response was detected in the tail artery in cholesterol-fed mice, and a significant correlation found between peak microvascular ACh response and maximum ACh response in the tail artery (r=0.699, P=0.017). No changes were found in SNP responses in the microvasculature or tail artery. Using this protocol, we have shown longitudinal decreases in microvascular endothelial function to cholesterol feeding. L-NAME studies confirm that the reduced vasodilatation to ACh in cholesterol-fed mice was mediated partly through reduced NO bioavailability. Wire myography of tail arteries confirmed that in-vivo measurements of microvascular function reflect ex-vivo vascular function in other beds. Longitudinal assessments of skin microvascular function in mice could provide a useful translatable model for assessing early endothelial dysfunction. PMID:23123637

Belch, Jill J F; Akbar, Naveed; Alapati, Venkateswara; Petrie, John; Arthur, Simon; Khan, Faisel

2012-10-31

145

In vivo Function and Membrane Binding Properties are Correlated for Escherichia coli LamB Signal Peptides  

NASA Astrophysics Data System (ADS)

Wild-type and pseudorevertant signal peptides of the lamB gene product of Escherichia coli interact with lipid systems whereas a nonfunctional deletion mutant signal peptide does not. This conclusion is based on (i) interaction of synthetic signal peptides with a lipid monolayer-water surface, (ii) conformational changes induced by presence of lipid vesicles in an aqueous solution of signal peptide, and (iii) capacities of the peptides to promote vesicle aggregation. Analysis of the signal sequences and previous conformational studies suggest that these lipid interaction properties may be attributable to the tendency of the functional signal peptides to adopt ? -helical conformations. Although the possibility of direct interaction between the signal peptide and membrane lipids during protein secretion is controversial, the results suggest that conformationally related amphiphilicity and consequent membrane affinity of signal sequences are important for function in vivo.

Briggs, Martha S.; Gierasch, Lila M.; Zlotnick, Adam; Lear, James D.; Degrado, William F.

1985-05-01

146

IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER  

PubMed Central

From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.

Eanes, Walter F.; Hey, Jody

1986-01-01

147

Dynamic contrast-enhanced optical imaging of in vivo organ function  

PubMed Central

Abstract. Conventional approaches to optical small animal molecular imaging suffer from poor resolution, limited sensitivity, and unreliable quantitation, often reducing their utility in practice. We previously demonstrated that the in vivo dynamics of an injected contrast agent could be exploited to provide high-contrast anatomical registration, owing to the temporal differences in each organ’s response to the circulating fluorophore. This study extends this approach to explore whether dynamic contrast-enhanced optical imaging (DyCE) can allow noninvasive, in vivo assessment of organ function by quantifying the differing cellular uptake or wash-out dynamics of an agent in healthy and damaged organs. Specifically, we used DyCE to visualize and measure the organ-specific uptake dynamics of indocyanine green before and after induction of transient liver damage. DyCE imaging was performed longitudinally over nine days, and blood samples collected at each imaging session were analyzed for alanine aminotransferase (ALT), a liver enzyme assessed clinically as a measure of liver damage. We show that changes in DyCE-derived dynamics of liver and kidney dye uptake caused by liver damage correlate linearly with ALT concentrations, with an r2 value of 0.91. Our results demonstrate that DyCE can provide quantitative, in vivo, longitudinal measures of organ function with inexpensive and simple data acquisition.

Amoozegar, Cyrus B.; Wang, Tracy; Bouchard, Matthew B.; McCaslin, Addason F. H.; Blaner, William S.; Levenson, Richard M.; Hillman, Elizabeth M. C.

2012-01-01

148

Intracardiac echocardiography: in vitro and in vivo validation for right ventricular volume and function.  

PubMed

To determine the feasibility and accuracy of intracardiac ultrasonography (ICUS) for the measurement of right ventricular (RV) volumes and function, a 10 MHz ICUS catheter was used in an in vitro and in vivo model. In the in vitro study, 16 sheep hearts were imaged. Sequential cross-sectional images from RV apex to base were recorded during a calibrated pullback. Volumes were calculated by applying Simpson's algorithm. ICUS-obtained volumes correlated well with actual volumes (standard error of estimate [SEE] = 2.3 ml, r = 0.98). For the in vivo study, a beating-heart canine model was used (31 hemodynamic stages in six dogs). Actual volumes were measured by an intracavitary balloon connected to an external column. Sequential cross-sectional images were recorded during the ICUS catheter pullback from apex to base of the RV, and volumes calculated by Simpson's algorithm. Good correlations were observed between ICUS and actual values for diastolic (SEE = 4.1 ml, r = 0.97), systolic (SEE = 3.4 ml, r = 0.96), and ejection fraction (SEE = 3.1%, r = 0.87) values. This new technique can accurately quantitate RV volumes, can function both in vitro and in vivo, and has the potential for increasing applications to questions of clinical and research interest. PMID:8579028

Vazquez de Prada, J A; Chen, M H; Guerrero, J L; Padial, L R; Jiang, L; Schwammenthal, E; Sagie, A; Weyman, A E; Levine, R A; Chen, C

1996-02-01

149

Differential Item Functioning Analysis Using Rasch Item Information Functions  

Microsoft Academic Search

Differential item functioning (DIF) analysis is a statistical technique used for ensuring the equity and fairness of educational assessments. This study formulates a new DIF analysis method using the information similarity index (ISI). ISI compares item information functions when data fits the Rasch model. Through simulations and an international assessment example, ISI, the signed area index (SAI), and Mantel-Haenszel procedure,

Adam E. Wyse; Raymond Mapuranga

2009-01-01

150

In vivo cardiac anatomical and functional effects of wheel running in mice by magnetic resonance imaging.  

PubMed

Physical activity is frequently used as a strategy to decrease pathogenesis and improve outcomes in chronic pathologies such as metabolic or cardiac diseases. In mice, it has been shown that voluntary wheel running (VWR) could induce an aerobic training effect and may provide a means of exploring the relationship between physical activity and the progression of pathology, or the effect of a drug on locomotor activity. To the best of our knowledge, in vivo magnetic resonance imaging (MRI) and other non-invasive methods had not been investigated for training evaluation in mice; therefore, it was proposed to test an MRI method coupled with a cardiorespiratory gating system on C57Bl/6 mice for in vivo heart anatomical and functional characterization in both trained and untrained animals. Twenty mice were either assigned to a 12-week VWR program or to a control group (CON - no wheel in the cage). At week 12, MRI scans showed an increase in the left ventricular (LV) wall mass in the VWR group compared with the CON group. The ex vivo measurements also found an increase in the heart and LV weight, as well as an increase in oxidative enzyme activities (i.e. cytochrome c oxidase [COx] in the soleus). In addition, correlations have been observed between ex vivo LV/body weight ratio, COx activity in the soleus and in vivo MRI LV wall mass/body weight. In conclusion, mouse cardiac MRI methods coupled with a cardio-respiratory gating system are sufficiently effective and feasible for non-invasive, training-induced heart hypertrophy characterization, and may be used for longitudinal training level follow-up in mouse models of cardiovascular and metabolic diseases. PMID:22328593

Aufradet, Emeline; Bessaad, Amine; Alsaid, Hasan; Schäfer, Florian; Sigovan, Monica; De Souza, Genevičve; Chirico, Erica; Martin, Cyril; Canet-Soulas, Emmanuelle

2012-02-10

151

Zebrafish primary testis tissue culture: an approach to study testis function ex vivo.  

PubMed

To develop new tools to study the regulation of testis physiology in teleost fish, a medium-term ex vivo organ culture system was adopted for zebrafish testis tissue. The addition of 100nM 11-ketotestosterone to the system supported complete spermatogenesis, as determined by morphological, molecular and immunohistochemical analyses. Under basal conditions, however, the development of differentiated spermatogonia, spermatocytes, and spermatids was seriously disturbed, probably related to the rapid (within 2 days) down-regulation of the steroidogenic system. Forskolin (0.5microM) stimulated acute androgen release from freshly removed tissue and partially prevented down-regulation of the steroidogenic system. The present ex vivo culture system can serve as a tool to evaluate effects of a wide range of substances on the two main functions of the testis, spermatogenesis and hormone production. PMID:19298819

Leal, Marcelo C; de Waal, Paul P; García-López, Angel; Chen, Shi X; Bogerd, Jan; Schulz, Rüdiger W

2009-03-17

152

Genetic Analysis of Myc and Telomerase Interactions In Vivo  

Microsoft Academic Search

Myc is a transcription factor with pleiotropic effects on tumorigenesis which are likely to be mediated by its target genes. A known Myc transcriptional target is the catalytic subunit of telomerase, Tert. However, the contribution of Tert activation to Myc-induced tumorigenesis in vivo remains unknown. In this study, we addressed the role of telomerase in Myc-induced skin papillomatosis by using

Ignacio Flores; Gerard Evan; M. A. Blasco

2006-01-01

153

Noninvasive Assessment of Gene Transfer and Expression by In Vivo Functional and Morphologic Imaging in a Rabbit Tumor Model  

PubMed Central

Purpose To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model. Materials and Methods Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, 111In-octreotide was administered intravenously after CT imaging using a clinical scanner. 111In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine 111In-octreotide biodistribution ex vivo. Results VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater 111In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r?=?0.72, P<0.01, Coefficient of the x-variable?=?.72) at 2 weeks than without excluding necrosis (P<0.01). Conclusion Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.

Ravoori, Murali K.; Han, Lin; Singh, Sheela P.; Dixon, Katherine; Duggal, Jyoti; Liu, Ping; Uthamanthil, Rajesh; Gupta, Sanjay; Wright, Kenneth C.; Kundra, Vikas

2013-01-01

154

40 CFR 798.5385 - In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis.  

Code of Federal Regulations, 2013 CFR

...ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) HEALTH EFFECTS TESTING GUIDELINES Genetic Toxicity § 798.5385 In vivo mammalian bone marrow cytogenetics tests: Chromosomal analysis. (a) Purpose....

2013-07-01

155

Relationship between in vivo activity and in vitro measures of function and stability of a protein  

SciTech Connect

The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein. 54 refs., 3 figs., 2 tabs.

Sandberg, W.S.; Schlunk, P.M.; Zabin, H.G. [Univ. of Chicago, IL (United States)] [and others

1995-09-19

156

Microfibril-associated Glycoprotein 2 (MAGP2) Loss of Function Has Pleiotropic Effects in Vivo.  

PubMed

Microfibril-associated glycoprotein (MAGP) 1 and 2 are evolutionarily related but structurally divergent proteins that are components of microfibrils of the extracellular matrix. Using mice with a targeted inactivation of Mfap5, the gene for MAGP2 protein, we demonstrate that MAGPs have shared as well as unique functions in vivo. Mfap5(-/-) mice appear grossly normal, are fertile, and have no reduction in life span. Cardiopulmonary development is typical. The animals are normotensive and have vascular compliance comparable with age-matched wild-type mice, which is indicative of normal, functional elastic fibers. Loss of MAGP2 alone does not significantly alter bone mass or architecture, and loss of MAGP2 in tandem with loss of MAGP1 does not exacerbate MAGP1-dependent osteopenia. MAGP2-deficient mice are neutropenic, which contrasts with monocytopenia described in MAGP1-deficient animals. This suggests that MAGP1 and MAGP2 have discrete functions in hematopoiesis. In the cardiovascular system, MAGP1;MAGP2 double knockout mice (Mfap2(-/-);Mfap5(-/-)) show age-dependent aortic dilation. These findings indicate that MAGPs have shared primary functions in maintaining large vessel integrity. In solid phase binding assays, MAGP2 binds active TGF?1, TGF?2, and BMP2. Together, these data demonstrate that loss of MAGP2 expression in vivo has pleiotropic effects potentially related to the ability of MAGP2 to regulate growth factors or participate in cell signaling. PMID:23963447

Combs, Michelle D; Knutsen, Russell H; Broekelmann, Thomas J; Toennies, Holly M; Brett, Thomas J; Miller, Chantel A; Kober, Daniel L; Craft, Clarissa S; Atkinson, Jeffrey J; Shipley, J Michael; Trask, Barbara C; Mecham, Robert P

2013-08-20

157

In Vivo Imaging of the Photoreceptor Mosaic in Retinal Dystrophies and Correlations with Visual Function  

PubMed Central

Purpose To relate in vivo microscopic retinal changes to visual function in patients who have various forms of retinal dystrophy. Methods The UC Davis Adaptive Optics (AO) fundus camera was used to acquire in vivo retinal images at the cellular level. Visual function tests consisting of visual fields, multifocal electroretinography (mfERG), and contrast sensitivity were measured in all subjects by using stimuli that were coincident with areas imaged. Five patients with different forms of retinal dystrophy and three control subjects were recruited. Cone densities were quantified for all retinal images. Results In all images of diseased retinas, there were extensive areas of dark space between groups of photoreceptors, where no cone photoreceptors were evident. These irregular features were not seen in healthy retinas, but were apparent in patients with retinal dystrophy. There were significant correlations between functional vision losses and the extent to which these irregularities, quantified by cone density, occurred in retinal images. Conclusions AO fundus imaging is a reliable technique for assessing and quantifying the changes in the photoreceptor layer as disease progresses. Furthermore, this technique can be useful in cases where visual function tests provide borderline or ambiguous results, as it allows visualization of individual photoreceptors.

Choi, Stacey S.; Doble, Nathan; Hardy, Joseph L.; Jones, Steven M.; Keltner, John L.; Olivier, Scot S.; Werner, John S.

2008-01-01

158

Spatiotemporal wavelet analysis for functional MRI  

Microsoft Academic Search

Characterizing the spatiotemporal behavior of the BOLD signal in functional Magnetic Resonance Imaging (fMRI) is a central issue in understanding brain function. While the nature of functional activation clusters is fundamentally heterogeneous, many current analysis approaches use spatially invariant models that can degrade anatomic boundaries and distort the underlying spatiotemporal signal. Furthermore, few analysis approaches use true spatiotemporal continuity in

Chris Long; Emery N. Brown; Dara Manoach; Victor Soloa

2004-01-01

159

PEG-Mediated Synthesis of Highly Dispersive Multifunctional Superparamagnetic Nanoparticles: Their Physicochemical Properties and Function In Vivo  

PubMed Central

Multifunctional superparamagnetic nanoparticles have been developed for a wide range of applications in nanomedicine, such as serving as tumor targeted drug carriers and molecular imaging agents. To function in vivo, the development of these novel materials must overcome several challenging requirements including biocompatibility, stability in physiological solutions, non-toxicity and the ability to traverse biological barriers. Here we report a PEG-mediated synthesis process to produce well-dispersed, ultrafine, and highly stable iron oxide nanoparticles for in vivo applications. Utilizing a biocompatible PEG coating bearing amine functional groups, the produced nanoparticles serve as an effective platform with the ability to incorporate a variety of targeting, therapeutic or imaging ligands. In this study, we demonstrated tumor-specific accumulation of these nanoparticles through both magnetic resonance and optical imaging after conjugation with chlorotoxin, a peptide with high affinity toward tumors of the neuroectodermal origin, and Cy5.5, a near-infrared fluorescent dye. Furthermore, we performed preliminary biodistribution and toxicity assessments of these nanoparticles in wild-type mice through histological analysis of clearance organs and hematology assay, and the results demonstrated the relative biocompatibility of these nanoparticles.

Sun, Conroy; Du, Kim; Fang, Chen; Bhattarai, Narayan; Veiseh, Omid; Kivit, Forrest; Stephen, Zachary; Lee, Donghoon; Ellenbogen, Richard G.; Ratner, Buddy; Zhang, Miqin

2010-01-01

160

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.  

PubMed Central

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.

Wang, J W; Wu, C F

1996-01-01

161

PEG-mediated synthesis of highly dispersive multifunctional superparamagnetic nanoparticles: their physicochemical properties and function in vivo.  

PubMed

Multifunctional superparamagnetic nanoparticles have been developed for a wide range of applications in nanomedicine, such as serving as tumor-targeted drug carriers and molecular imaging agents. To function in vivo, the development of these novel materials must overcome several challenging requirements including biocompatibility, stability in physiological solutions, nontoxicity, and the ability to traverse biological barriers. Here we report a PEG-mediated synthesis process to produce well-dispersed, ultrafine, and highly stable iron oxide nanoparticles for in vivo applications. Utilizing a biocompatible PEG coating bearing amine functional groups, the produced nanoparticles serve as an effective platform with the ability to incorporate a variety of targeting, therapeutic, or imaging ligands. In this study, we demonstrated tumor-specific accumulation of these nanoparticles through both magnetic resonance and optical imaging after conjugation with chlorotoxin, a peptide with high affinity toward tumors of the neuroectodermal origin, and Cy5.5, a near-infrared fluorescent dye. Furthermore, we performed preliminary biodistribution and toxicity assessments of these nanoparticles in wild-type mice through histological analysis of clearance organs and hematology assay, and the results demonstrated the relative biocompatibility of these nanoparticles. PMID:20232826

Sun, Conroy; Du, Kim; Fang, Chen; Bhattarai, Narayan; Veiseh, Omid; Kievit, Forrest; Stephen, Zachary; Lee, Donghoon; Ellenbogen, Richard G; Ratner, Buddy; Zhang, Miqin

2010-04-27

162

Applications of nuclear technologies for in-vivo elemental analysis  

SciTech Connect

Measurement facilities developed, to date, include a unique whole-body-counter, (WBC); a total-body neutron-activation facility (TBNAA); and a partial-body activation facility (PBNAA). A variation of the prompt-gamma neutron-activation technique for measuring total-body nitrogen was developed to study body composition of cancer patients and the effect of nutritional regimens on the composition. These new techniques provide data in numerous clinical studies not previously amenable to investigation. The development and perfection of these techniques provide unique applications of radiation and radioisotopes to the early diagnosis of certain diseases and the evaluation of therapeutic programs. The PBNAA technique has been developed and calibrated for in-vivo measurement of metals. Development has gone forward on prompt-gamma neutron activation for the measurement of cadmium, x-ray fluorescence (XRF) for measurement of iron. Other techniques are being investigated for in-vivo measurement of metals such as silicon and beryllium.

Cohn, S.H.; Ellis, K.J.; Vartsky, D.; Wielopolski, L.

1982-01-01

163

Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding.  

PubMed

Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering. PMID:24152813

Qiu, Juhui; Zheng, Yiming; Hu, Jianjun; Liao, Donghua; Gregersen, Hans; Deng, Xiaoyan; Fan, Yubo; Wang, Guixue

2013-10-23

164

Brain functional networks analysis and comparison  

Microsoft Academic Search

This study used the complex network analysis to examine the brain functional network involved in right finger movements and compare the deferent functional network involved in left finger and right finger movements. We found that (a)the connections change exponentially as distance between nodes change, the function is Gaussian; (b) the distribution of functional connections was scale-free; (c) the typical path

Fangfeng Zhang; Chunhui Chen; Lu Jiang

2010-01-01

165

[In vivo single molecular fluorescence imaging for analysis of pharmacokinetics].  

PubMed

Nano-materials are expected for research on molecular imaging of pharmacokinetics. We measured in vivo migration of CdSe nano-particles(Quantum Dots(QDs))conjugated with monoclonal anti-HER2 antibody(trastuzumab)in tumor vessel to breast cancer cells. We established a high resolution in vivo 3D microscopic system for a novel imaging method at single molecular level. The HER2 protein expressed in cancer cells and its dynamics were visualized by QDs in vivo at the spatial resolution of 30 nm. It suggests future utilization of the system in medical applications to improve the drug delivery system to target primary and metastatic tumors for made-to-order treatment. Future innovation in cancer imaging by nano-technology and novel measurement technology will provide great improvement, not only in the clinical field, but also in basic medical science. Advances in nano-biotechnology have great potential to improve prevention, diagnosis and treatment of human disease. PMID:18701837

Takeda, Motohiro; Gonda, Kohsuke; Higuchi, Hideo; Ohuchi, Noriaki

2008-08-01

166

Fatigue alters in vivo function within and between limb muscles during locomotion  

PubMed Central

Muscle fatigue, a reduction in force as a consequence of exercise, is an important factor for any animal that moves, and can result from both peripheral and/or central mechanisms. Although much is known about whole-limb force generation and activation patterns in fatigued muscles under sustained isometric contractions, little is known about the in vivo dynamics of limb muscle function in relation to whole-body fatigue. Here we show that limb kinematics and contractile function in the lateral (LG) and medial (MG) gastrocnemius of helmeted guineafowl (Numida meleagris) are significantly altered following fatiguing exercise at 2?m?s?1 on an inclined treadmill. The two most significant findings were that the variation in muscle force generation, measured directly from the muscles' tendons, increased significantly with fatigue, and fascicle shortening in the proximal MG, but not the distal MG, decreased significantly with fatigue. We suggest that the former is a potential mechanism for decreased stability associated with fatigue. The region-specific alteration of fascicle behaviour within the MG as a result of fatigue suggests a complex response to fatigue that probably depends on muscle–aponeurosis and tendon architecture not previously explored. These findings highlight the importance of studying the integrative in vivo dynamics of muscle function in response to fatigue.

Higham, Timothy E.; Biewener, Andrew A.

2008-01-01

167

Functional Brain Image Analysis Using Joint Function-Structure Priors  

Microsoft Academic Search

\\u000a We propose a new method for context-driven analysis of functional magnetic resonance images (fMRI) that incorporates spatial\\u000a relationships between functional parameter clusters and anatomical structure directly for the first time. We design a parametric\\u000a scheme that relates functional and structural spatially-compact regions in a single unified manner. Our method is motivated\\u000a by the fact that the fMRI and anatomical MRI

Jing Yang; Xenophon Papademetris; Lawrence H. Staib; Robert T. Schultz; James S. Duncan

2004-01-01

168

Singularity Analysis of Generating Functions  

Microsoft Academic Search

This work presents a class of methods by which one can translate, on a term-by-term basis, an asymptotic expansion ofa function around a dominant singularity into a corresponding asymptotic expansion for the Taylor coefficients ofthe function. This approach is based on contour integration using Cauchy's formula and Hankel-like contours. It constitutes an alternative to either Darboux's method or Tauberian theorems

Philippe Flajolet; Andrew M. Odlyzko

1990-01-01

169

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function.  

PubMed

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis. PMID:23236142

Rodriguez-Diaz, Rayner; Speier, Stephan; Molano, Ruth Damaris; Formoso, Alexander; Gans, Itai; Abdulreda, Midhat H; Cabrera, Over; Molina, Judith; Fachado, Alberto; Ricordi, Camillo; Leibiger, Ingo; Pileggi, Antonello; Berggren, Per-Olof; Caicedo, Alejandro

2012-12-10

170

Noninvasive in vivo model demonstrating the effects of autonomic innervation on pancreatic islet function  

PubMed Central

The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.

Rodriguez-Diaz, Rayner; Speier, Stephan; Molano, Ruth Damaris; Formoso, Alexander; Gans, Itai; Abdulreda, Midhat H.; Cabrera, Over; Molina, Judith; Fachado, Alberto; Ricordi, Camillo; Leibiger, Ingo; Pileggi, Antonello; Berggren, Per-Olof; Caicedo, Alejandro

2012-01-01

171

Numerical and In Vivo Validation of Fast Cine DENSE MRI for Quantification of Regional Cardiac Function  

PubMed Central

Quantitative assessment of regional cardiac function can improve the accuracy of detecting wall motion abnormalities due to heart disease. While recently developed fast cine displacement-encoded with stimulated echoes (DENSE) MRI is a promising modality for the quantification of regional myocardial function, it has not been validated for clinical applications. The purpose of this study, therefore, was to validate the accuracy of fast cine DENSE MRI with numerical simulation and in vivo experiments. A numerical phantom was generated to model physiologically relevant deformation of the heart, and the accuracy of fast cine DENSE was evaluated against the numerical reference. For in vivo validation, 12 controls and 13 heart disease patients were imaged using both fast cine DENSE and myocardial tagged MRI. Numerical simulation demonstrated that the echo-combination DENSE reconstruction method is relatively insensitive to clinically relevant resonance frequency offsets. The strain measurements by fast cine DENSE and the numerical reference were strongly correlated and in excellent agreement (mean difference=0.00; 95% limits of agreement were 0.01 and ?0.02). The strain measurements by fast cine DENSE and myocardial tagged MRI were strongly correlated (correlation coefficient = 0.92) and in good agreement (mean difference=0.01; 95% limits of agreement were 0.07 and ?0.04).

Feng, Li; Donnino, Robert; Babb, James; Axel, Leon; Kim, Daniel

2009-01-01

172

Tumor regression in vivo by photothermal therapy based on gold-nanorod-loaded, functional nanocarriers.  

PubMed

We developed a very effective hyperthermia system for successful photothermal cancer therapy. Instead of applying individual gold nanorods (GNRs) that can absorb NIR light, GNRs were loaded into functional nanocarriers that could provide stable storage of GNRs and selective delivery to a target tumor site. The functional nanocarriers (chitosan-conjugated, Pluronic-based nanocarriers) were prepared by chemically cross-linking Pluronic F 68 with chitosan conjugation to form a flexible, soft, and excellent reservoir for biomacromolecules as well as tumor targeting. In vivo characteristics of the nanocarriers including a long circulation time, a good tumor accumulation, and low liver uptake were previously characterized by us. When GNRs were delivered by using these nanocarriers, much enhanced in vitro cellular uptake and a photothermal effect were observed for a cancer cell line. More importantly, an intravenous injection of this system followed by NIR laser irradiation to the tumor site resulted in a very efficient thermolysis in vivo. Thus, apparently complete tumor resorption was achieved without damage to the surrounding tissue, suggesting a promising candidate for clinical phototherapeutic applications. PMID:21344891

Choi, Won Il; Kim, Ja-Young; Kang, Chul; Byeon, Clare C; Kim, Young Ha; Tae, Giyoong

2011-02-23

173

In vivo and in vitro HeNe laser effects on phagocyte functions  

SciTech Connect

The goal of this work was to evaluate the effect of helium-neon (HeNe) laser irradiation on immunocompetent cells. We used the in vivo skin window method and in vitro granulocyte function tests. The study of cellular migration showed a marked decrease in vitro and in vivo in a dose-independent manner. Superoxide release was not modified by laser irradiation. The granulocyte's aggregation, when using PHA and PMA, presented a reduction that was statistically very significant, not as a subordinate dose. An increase of the release of ATP was demonstrated only at 4 joules and precedes granulocyte aggregation. When using Ca2+ ionophore A23187 as stimulus, laser irradiation at 1, 2 or 4J did not show any modification of granulocyte aggregation. The monoclonal antibody 60.1, which identifies a membrane antigen fundamental for aggregation and chemotaxis, is expressed in normal amounts on granulocyte membranes both before and after irradiation with a HeNe laser. In fact, laser irradiation preferentially attacks the area of the cellular centrosome that determines a modification of cellular morphology. The electron microscope and immunofluorescence study with a monoclonal antibody have pointed out a disorganization of the microtubules. The alteration of some of the granulocyte functions is correlated to the damage in the centrioles. The granulocyte mitochondrial system and surface membrane remain intact, and this explains the normal production and release of free radicals. Further experiments are necessary to evaluate the clinical application of lasers in various diseases with immunophagocytic pathogenesis.

Ricevuti, G.; Mazzone, A.; Monaia, C.; Fratino, P.; Degiulio, R.; Dell'Acqua, R.; Leonardi, G.; Jucci, A.; Sacchi, S. (Univ. of Pavia (Italy))

1989-10-01

174

Tartary buckwheat improves cognition and memory function in an in vivo amyloid-?-induced Alzheimer model.  

PubMed

Protective effects of Tartary buckwheat (TB) and common buckwheat (CB) on amyloid beta (A?)-induced impairment of cognition and memory function were investigated in vivo in order to identify potential therapeutic agents against Alzheimer's disease (AD) and its associated progressive memory deficits, cognitive impairment, and personality changes. An in vivo mouse model of AD was created by injecting the brains of ICR mice with A?(25-35), a fragment of the full-length A? protein. Damage of mice recognition ability through following A?(25-35) brain injections was confirmed using the T-maze test, the object recognition test, and the Morris water maze test. Results of behavior tests in AD model showed that oral administration of the methanol (MeOH) extracts of TB and CB improved cognition and memory function following A?(25-35) injections. Furthermore, in groups receiving the MeOH extracts of TB and CB, lipid peroxidation was significantly inhibited, and nitric oxide levels in tissue, which are elevated by injection of A?(25-35), were also decrease. In particular, the MeOH extract of TB exerted a stronger protective activity than CB against A?(25-35)-induced memory and cognition impairment. The results indicate that TB may play a promising role in preventing or reversing memory and cognition loss associated with A?(25-35)-induced AD. PMID:23219778

Choi, Ji Yeon; Cho, Eun Ju; Lee, Hae Song; Lee, Jeong Min; Yoon, Young-Ho; Lee, Sanghyun

2012-11-28

175

An In Vivo Functional Screen Uncovers miR-150-Mediated Regulation of Hematopoietic Injury Response  

PubMed Central

Summary Hematopoietic stem and progenitor cells are often undesired targets of chemotherapies, leading to hematopoietic suppression requiring careful clinical management. Whether microRNAs control hematopoietic-injury response is largely unknown. We report a novel in vivo gain-of-function screen and identification of miR-150 as an inhibitor of hematopoietic recovery upon 5-fluorouracil-induced injury. Utilizing a bone marrow transplant model with a barcoded microRNA-library, we screened for barcode abundance in peripheral blood of recipient mice before and after 5-fluorouracil treatment. Overexpression of screen-candidate miR-150 resulted in significantly slowed recovery rates across major blood lineages, with associated impairment of bone marrow clonogenic potential. Conversely, platelets and myeloid cells from miR-150-null marrow recovered faster after 5-fluorouracil treatment. Heterozygous knockout of c-myb, a conserved target of miR-150, partially phenocopied miR-150 forced expression. Our data highlight the role of microRNAs in controlling hematopoietic-injury response, and demonstrate the power of in vivo functional screens for studying microRNAs in normal tissue physiology.

Adams, Brian D.; Guo, Shangqin; Bai, Haitao; Guo, Yanwen; Megyola, Cynthia; Cheng, Jijun; Heydari, Kartoosh; Xiao, Changchun; Reddy, E. Premkumar; Lu, Jun

2012-01-01

176

Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins  

Microsoft Academic Search

14-3-3 proteins play a role in many cellular functions: they bind to and regulate several proteins which are critical for cell proliferation and differentiation. 14-3-3 proteins exist as dimers, and in this study we have shown that diverse 14-3-3 proteins can form both homo- and heterodimers in vitro (by crosslinking studies) and in vivo (by coimmunoprecipitation and Western blot analysis);

David H. Jones; Steven Ley; Alastair Aitken

1995-01-01

177

The archetypal R90C CADASIL-NOTCH3 mutation retains NOTCH3 function in vivo.  

PubMed

Cerebral Autosomal Dominant Arteriopathy with Subcortical infarcts and Leukoencephalopathy (CADASIL) is the most prominent known cause of inherited stroke and vascular dementia in human adult. The disease gene, NOTCH3, encodes a transmembrane receptor primarily expressed in arterial smooth muscle cells (SMC). Pathogenic mutations lead to an odd number of cysteine residues within the NOTCH3 extracellular domain (NOTCH3(ECD)), and are associated with progressive accumulation of NOTCH3(ECD) at the SMC plasma membrane. The murine homolog, Notch3, is dispensable for viability but required post-natally for the elaboration and maintenance of arteries. How CADASIL-associated mutations impact NOTCH3 function remains a fundamental, yet unresolved issue. Particularly, whether NOTCH3(ECD) accumulation may titrate the ligand and inhibit the normal pathway is unknown. Herein, using genetic analyses in the mouse, we assessed the functional significance of an archetypal CADASIL-associated mutation (R90C), in vivo, in brain arteries. We show that transgenic mouse lines expressing either the wild-type human NOTCH3 or the mutant R90C human NOTCH3, at comparable and physiological levels, can rescue the arterial defects of Notch3-/- mice to similar degrees. In vivo assessment of NOTCH3/RBP-Jk activity provides evidence that the mutant NOTCH3 protein exhibits normal level of activity in brain arteries. Remarkably, the mutant NOTCH3 protein remains functional and does not exhibit dominant negative interfering activity, even when NOTCH3(ECD) accumulates. Collectively, these data suggest a model that invokes novel pathogenic roles for the mutant NOTCH3 protein rather than compromised NOTCH3 function as the primary determinant of the CADASIL arteriopathy. PMID:17331978

Monet, Marie; Domenga, Valérie; Lemaire, Barbara; Souilhol, Céline; Langa, Francina; Babinet, Charles; Gridley, Thomas; Tournier-Lasserve, Elisabeth; Cohen-Tannoudji, Michel; Joutel, Anne

2007-03-01

178

In vivo elemental analysis by counting neutron-induced gamma rays for medical and biological applications  

Microsoft Academic Search

Non-invasive in vivo elemental analysis is a technique used to assess human body composition which is indicative of nutritional status and health condition. The in vivo measurement of the body's major elements is used for a variety of medical studies requiring the determination of the body's compartments (protein, fat, water, bone). Whole body gamma-ray counters, consisting of Nal(Tl) crystal detectors

Joseph J. Kehayias; Ruimei Ma; Hong Zhuang; Robert Moore; Lisa Dowling

1995-01-01

179

Software benchmarks using function point analysis  

Microsoft Academic Search

The alternative to using source lines of code (SLOC) for costing software projects is to use function points. Functional point analysis (FPA), which was first introduced in 1979, has now been widely accepted as the industry standard for estimating software size and costs. International standard bodies like the International Function Point Users’ Group (IFPUG) has been maintaining a repository of

Yen Cheung; Rob Willis; Barrie Milne

1999-01-01

180

Functional analysis and treatment of diurnal bruxism.  

PubMed

An analogue functional analysis identified attention as a function for a 5-year-old boy's bruxism (teeth grinding). Functional communication training resulted in a reduction of bruxism and an increase in alternative mands for attention. Results were maintained 3 weeks following the intervention. PMID:24114107

Lang, Russell; Davenport, Katy; Britt, Courtney; Ninci, Jennifer; Garner, Jennifer; Moore, Melissa

2013-02-20

181

In-vivo neutron activation analysis: principles and clinical applications  

SciTech Connect

In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into and modelling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, enhancing uniformity, and reducing the dose required for the measurement. The work presently underway will yield significant data on a variety of environmental contaminants such as Cd. Compositional studies are determining the level of vital constituents such as nitrogen and potassium in both normal subjects and in patients with a variety of metabolic disorders. Therapeutic programs can be assessed while in progress. It seems likely that by the end of this century there will have been significant progress with this research tool, and exciting insights obtained into the nature and dynamics of human body composition.

Cohn, S.H.

1982-01-01

182

Clinical applications of in vivo neutron-activation analysis  

SciTech Connect

In vivo neutron activation has opened a new era of both clinical diagnosis and therapy evaluation, and investigation into and modelling of body composition. The techniques are new, but it is already clear that considerable strides can be made in increasing accuracy and precision, increasing the number of elements susceptible to measurement, enhancing uniformity, and reducing the dose required for the measurement. The work presently underway will yield significant data on a variety of environmental contaminants such as Cd. Compositional studies are determining the level of vital constituents such as nitrogen and potassium in both normal subjects and in patients with a variety of metabolic disorders. Therapeutic programs can be assessed while in progress.

Cohn, S.H.

1982-01-01

183

Synaptic Structure and Function in the Mouse Somatosensory Cortex during Chronic Pain: In Vivo Two-Photon Imaging  

PubMed Central

Recent advances in two-photon microscopy and fluorescence labeling techniques have enabled us to directly see the structural and functional changes in neurons and glia, and even at synapses, in the brain of living animals. Long-term in vivo two-photon imaging studies have shown that some postsynaptic dendritic spines in the adult cortex are rapidly eliminated or newly generated, in response to altered sensory input or synaptic activity, resulting in experience/activity-dependent rewiring of neuronal circuits. In vivo Ca2+ imaging studies have revealed the distinct, input-specific response patterns of excitatory neurons in the brain. These updated in vivo approaches are just beginning to be used for the study of pathophysiological mechanisms of chronic diseases. In this paper, we introduce recent in vivo two-photon imaging studies demonstrating how plastic changes in synaptic structure and function of the mouse somatosensory cortex, following peripheral injury, contribute to chronic pain conditions, like neuropathic and inflammatory pain.

Kim, Sun Kwang; Eto, Kei; Nabekura, Junichi

2012-01-01

184

Characterization of the structural and functional determinants of MANF/CDNF in Drosophila in vivo model.  

PubMed

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo. PMID:24019940

Lindström, Riitta; Lindholm, Päivi; Kallijärvi, Jukka; Yu, Li-Ying; Piepponen, T Petteri; Arumäe, Urmas; Saarma, Mart; Heino, Tapio I

2013-09-03

185

Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo.  

PubMed

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17?-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 ± 11.8%, OGD-estrogen: 102.6 ± 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17?-estradiol (OGD-estrogen-G15: 89.5 ± 6.9, P < 0.05 compared with 17?-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17?-estradiol (BUN/SCr 17?-estradiol: 34 ± 19/0.2 ± 0.1 vehicle: 92 ± 49/0.5 ± 0.3, n = 8-12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17?-estradiol treatment (?; 17?-estradiol: 0.74 ± 0.26 vs. vehicle: 1.05 ± 0.53, n = 14-15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo. PMID:22622457

Hutchens, Michael P; Fujiyoshi, Tetsuhiro; Komers, Radko; Herson, Paco S; Anderson, Sharon

2012-05-23

186

Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo  

PubMed Central

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17?-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 ± 11.8%, OGD-estrogen: 102.6 ± 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17?-estradiol (OGD-estrogen-G15: 89.5 ± 6.9, P < 0.05 compared with 17?-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17?-estradiol (BUN/SCr 17?-estradiol: 34 ± 19/0.2 ± 0.1 vehicle: 92 ± 49/0.5 ± 0.3, n = 8–12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17?-estradiol treatment (?; 17?-estradiol: 0.74 ± 0.26 vs. vehicle: 1.05 ± 0.53, n = 14–15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo.

Fujiyoshi, Tetsuhiro; Komers, Radko; Herson, Paco S.; Anderson, Sharon

2012-01-01

187

Exoribonuclease and endoribonuclease activities of RNase BN/RNase Z both function in vivo.  

PubMed

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli. PMID:22893707

Dutta, Tanmay; Malhotra, Arun; Deutscher, Murray P

2012-08-14

188

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo*  

PubMed Central

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

Dutta, Tanmay; Malhotra, Arun; Deutscher, Murray P.

2012-01-01

189

Characterization of the Structural and Functional Determinants of MANF/CDNF in Drosophila In Vivo Model  

PubMed Central

Mammalian MANF and CDNF proteins are evolutionarily conserved neurotrophic factors that can protect and repair mammalian dopaminergic neurons in vivo. In Drosophila, the sole MANF protein (DmManf) is needed for the maintenance of dopaminergic neurites and dopamine levels. Although both secreted and intracellular roles for MANF and CDNF have been demonstrated, very little is known about the molecular mechanism of their action. Here, by using a transgenic rescue approach in the DmManf mutant background we show that only full-length MANF containing both the amino-terminal saposin-like and carboxy-terminal SAP-domains can rescue the larval lethality of the DmManf mutant. Independent N- or C-terminal domains of MANF, even when co-expressed together, fail to rescue. Deleting the signal peptide or mutating the CXXC motif in the C-terminal domain destroys the activity of full-length DmManf. Positively charged surface amino acids and the C-terminal endoplasmic reticulum retention signal are necessary for rescue of DmManf mutant lethality when DmManf is expressed in a restricted pattern. Furthermore, rescue experiments with non-ubiquitous expression reveals functional differences between the C-terminal domain of human MANF and CDNF. Finally, DmManf and its C-terminal domain rescue mammalian sympathetic neurons from toxin-induced apoptosis in vitro demonstrating functional similarity of the mammalian and fly proteins. Our study offers further insights into the functional conservation between invertebrate and mammalian MANF/CDNF proteins and reveals the importance of the C-terminal domain for MANF activity in vivo.

Lindstrom, Riitta; Lindholm, Paivi; Kallijarvi, Jukka; Yu, Li-ying; Piepponen, T. Petteri; Arumae, Urmas; Saarma, Mart; Heino, Tapio I.

2013-01-01

190

MS-based metabolomics facilitates the discovery of in vivo functional small molecules with a diversity of biological contexts.  

PubMed

In vivo small molecules as necessary intermediates are involved in numerous critical metabolic pathways and biological processes associated with many essential biological functions and events. There is growing evidence that MS-based metabolomics is emerging as a powerful tool to facilitate the discovery of functional small molecules that can better our understanding of development, infection, nutrition, disease, toxicity, drug therapeutics, gene modifications and host-pathogen interaction from metabolic perspectives. However, further progress must still be made in MS-based metabolomics because of the shortcomings in the current technologies and knowledge. This technique-driven review aims to explore the discovery of in vivo functional small molecules facilitated by MS-based metabolomics and to highlight the analytic capabilities and promising applications of this discovery strategy. Moreover, the biological significance of the discovery of in vivo functional small molecules with different biological contexts is also interrogated at a metabolic perspective. PMID:24175746

Yan, Leyu; Nie, Wenna; Parker, Tony; Upton, Zee; Lu, Haitao

2013-10-01

191

Optogenetic analysis of synaptic function  

Microsoft Academic Search

We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or ?-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for ?-aminobutyric acid) or contraction (for acetylcholine), which we analyzed

Jana F Liewald; Martin Brauner; Greg J Stephens; Magali Bouhours; Christian Schultheis; Mei Zhen; Alexander Gottschalk

2008-01-01

192

Energy function analysis for power system stability  

Microsoft Academic Search

Energy Function Analysis for Power System Stability presents the concept of energy function, which has found wide-spread applications for power systems in recent years. The most recent advances in five distinct areas are reviewed: Development of energy functions for structure preserving models, which can incorporate non-linear load models; energy functions which include a detailed model of the generating unit (i.e.

M. A. Pai

1989-01-01

193

Automating Functional Harmonic Analysis: The Funchal System  

Microsoft Academic Search

Functional harmonic analysis is an important task in music composition, accompaniment, arrangement and others. However, the solutions are still not satisfactory. The proposed process is divided into two levels: the former extends one of previous works in the domain to carry out a richer analysis of chord grids and is where the very analysis is performed, and the latter is

Ricardo Scholz; Vítor Dantas; Geber Ramalho

2005-01-01

194

Elevation of transcription factor Islet-1 levels in vivo increases ?-cell function but not ?-cell mass.  

PubMed

A decrease in the expression of Islet-1 (Isl-1), an islet transcription factor, has been reported in several physiological settings of reduced ?-cell function. Here, we investigate whether an increased level of Isl-1 in islet cells can enhance ?-cell function and/or mass. We demonstrate that transgenic mice with Isl-1 overexpression display improved glucose tolerance and enhanced insulin secretion without significant changes in ? cell mass. From our microarray study, we identify approximately 135 differentially expressed genes in the islets of Isl-1 overexpressing mice that have been implicated to function in numerous biological processes including protein trafficking, metabolism and differentiation. Using real-time PCR we have confirmed upregulation of Caps2, Sec14l4, Slc2a10, P2rx7, Afamin, and Neurogenin 3 that may in part mediate the observed improved insulin secretion in Isl-1 overexpressing mice. These findings show for the first time that Isl-1 is a key factor in regulating adult ? cell function in vivo, and suggest that Isl-1 elevation could be beneficial to improve glucose homeostasis. PMID:22595886

Liu, Jingxuan; Walp, Erik R; May, Catherine Lee

2012-05-01

195

Imaging the Function of P-Glycoprotein With Radiotracers: Pharmacokinetics and In Vivo Applications  

PubMed Central

P-glycoprotein (P-gp), an efflux transporter, controls the pharmacokinetics of various compounds under physiological conditions. P-gp-mediated drug efflux has been suggested as playing a role in various disorders, including multidrug-resistant cancer and medication-refractory epilepsy. However, P-gp inhibition has had, to date, little or no clinically significant effect in multidrug-resistant cancer. To enhance our understanding of its in vivo function under pathophysiological conditions, substrates of P-gp have been radiolabeled and imaged using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). To accurately quantify P-gp function, a radiolabeled P-gp substrate should be selective for P-gp, produce a large signal after P-gp blockade, and generate few radiometabolites that enter the target tissue. Furthermore, quantification of P-gp function via imaging requires pharmacological inhibition of P-gp, which requires knowledge of P-gp density at the target site. By meeting these criteria, imaging can elucidate the function of P-gp in various disorders and improve the efficacy of treatments.

Kannan, P; John, C; Zoghbi, SS; Halldin, C; Gottesman, MM; Innis, RB; Hall, MD

2009-01-01

196

Cellular in vivo imaging reveals coordinated regulation of pituitary microcirculation and GH cell network function.  

PubMed

Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation. PMID:20160103

Lafont, Chrystel; Desarménien, Michel G; Cassou, Mathieu; Molino, François; Lecoq, Jérôme; Hodson, David; Lacampagne, Alain; Mennessier, Gérard; El Yandouzi, Taoufik; Carmignac, Danielle; Fontanaud, Pierre; Christian, Helen; Coutry, Nathalie; Fernandez-Fuente, Marta; Charpak, Serge; Le Tissier, Paul; Robinson, Iain C A F; Mollard, Patrice

2010-02-16

197

Characterization of antisera against bovine prolactin for in vivo studies on prolactin function in the rat.  

PubMed Central

The IgG fraction of rabbit antisera to bovine prolactin (PRL), intended for in vivo studies on the role of PRL in the rat, was prepared and characterized in vitro and in vivo. The antibodies showed a strong reaction with bovine PRL in double diffusion, immunoelectrophoresis, radioimmunoassay and passive haemagglutination using bovine PRL-coated erythrocytes. In indirect immunofluorescence on paraffin sections of bovine pituitary glands the antibodies could be used for the detection of PRL-producing cells. Cross-reaction with rat PRL was observed in passive haemagglutination with rat PRL-coated erythrocytes and in indirect immunofluorescence on rat pituitary gland, but not in any of the other test systems. The ability of the antibodies to neutralize homologous, i.e. bovine, PRL was tested in lactating rats depleted of endogenous PRL by bromergocriptin treatment. The impaired lactation performance of such animals can be restored by substitution with bovine PRL. If the bovine PRL used for substitution was complexed with anti-bovine PRL-IgG, it lost its biological activity. On the other hand, injections of even high amounts of the antibodies into lactating rats failed to reveal any effect on lactation. It is concluded that either the antibodies do not cross-react with circulating rat PRL in contrast to pituitary PRL (preprolactin?) or that the cross-reacting antibody-populations(s) lack(s) the ability to neutralize the biological function of rat PRL.

Kofler, R; Tabarelli, M; Schwarz, S; Wolf, H; Loewit, K; Wick, G

1980-01-01

198

In vitro hematological and in vivo vasoactivity assessment of dextran functionalized graphene.  

PubMed

The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ? 7?mg/ml, showed a 12-20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1-50?mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect. PMID:24002570

Chowdhury, Sayan Mullick; Kanakia, Shruti; Toussaint, Jimmy D; Frame, Mary D; Dewar, Anthony M; Shroyer, Kenneth R; Moore, William; Sitharaman, Balaji

2013-09-01

199

In Vivo Evaluation of Vena Caval Filters: Can Function Be Linked to Design Characteristics?  

SciTech Connect

Purpose: To compare the five vena caval filters marketed in the United States and one investigational vena caval filter and to determine whether there is an association between their design and their in vivo function.Methods: Four of each type of filter-Simon Nitinol (SN), Bird's Nest (BN), Vena Tech (VT), Greenfield stainless steel (PSGF), Greenfield titanium (TGF), and the investigational stent cone filter (NGF)-were studied for 60 days in 12 sheep. Radiographic and pathologic outcomes to be assessed included clot capture and resolution, vena caval penetration, position of the filter, thrombogenicity, and vessel wall reaction.Results: Filters differed with respect to the number of clot-trapping levels and the interdependence of the legs. All devices were successfully placed. Intentionally embolized clot was captured. One VT and two SN filters migrated in response to clot capture. Resolution of thrombus was variable, and related to the design of the device. Fibrin webbing was widely present with the VT, BN, and SN filters but limited in the others. The VT and NGF filters demonstrated the most stable filter base diameter.Conclusions: The performance of vena caval filters differs with respect to clot resolution and mechanical stability. Interdependent filter limbs and single-stage conical capture sites appear to result in more favorable performance in in vivo studies.

Proctor, Mary C. [Department of Surgery, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States); Cho, Kyung J. [Department of Radiology, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States); Greenfield, Lazar J. [Department of Surgery, University of Michigan Hospitals, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0346 (United States)

2000-11-15

200

Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes  

PubMed Central

The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called “nanophotothermolysis”. We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion.

Mocan, Lucian; Tabaran, Flaviu A; Mocan, Teodora; Bele, Constantin; Orza, Anamaria Ioana; Lucan, Ciprian; Stiufiuc, Rares; Manaila, Ioana; Iulia, Ferencz; Dana, Iancu; Zaharie, Florin; Osian, Gelu; Vlad, Liviu; Iancu, Cornel

2011-01-01

201

Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes.  

PubMed

The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called "nanophotothermolysis". We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion. PMID:21720504

Mocan, Lucian; Tabaran, Flaviu A; Mocan, Teodora; Bele, Constantin; Orza, Anamaria Ioana; Lucan, Ciprian; Stiufiuc, Rares; Manaila, Ioana; Iulia, Ferencz; Dana, Iancu; Zaharie, Florin; Osian, Gelu; Vlad, Liviu; Iancu, Cornel

2011-04-28

202

Doxycycline's Effect on Ocular Angiogenesis: an In Vivo Analysis  

PubMed Central

Purpose To determine the in vivo effect of doxycycline (doxy) on choroidal angiogenesis and pterygium growth by using a choroidal neovascular murine model (CNV), a directed in vivo angiogenesis assay (DIVAA) and a pterygium murine model. Design Experimental Study Participants 3 murine models were investigated with 4 mice minimum per group and 22 maximum per group. Methods Mice received water with or without doxycycline (Leiter's Pharmacy, San Jose, CA). For the CNV, the neovascular lesion volume was determined in choroid-retinal pigment epithelial (RPE) flat mounts using confocal microscopy seven days after laser induction. For DIVAA, silicone capsules containing 10,000 human pterygium epithelial cells were implanted in the flanks of mice subcutaneously. After eleven days, neovascularization (NV) was quantified using spectrofluorimetry after murine tail-vein injection of fluorescein isothiocyanate (FITC)-labeled dextran. A pterygium epithelial cell model was developed by injecting 10,000 human pterygium epithelial cells in the nasal subconjunctival space in athymic nude mice. Doxy was started on day six at 50 mg/kg/day; corneal lesions that resulted from the injections were compared at days six and fifteen. Main outcome measures Student's t-test was used to evaluate the data for the CNV and DIVAA models and histologic preparations were used to evaluate pterygia lesions. Results There was significantly less NV and lesion volume with doxy taken in drinking water versus plain water. With doxy treatment, the laser-induced CNV showed a maximal 66% decrease in choroidal blood vessel volume (p?0.008) and the DIVAA showed a 30% reduction of blood vessel growth and migration (p<0.004). Histologic preparations demonstrated that pterygium cell lesions regressed when mice were administered doxy for 9 days. Conclusions Doxycycline significantly inhibited angiogenesis in three murine models. The most dramatic effect was found in the choroidal neovascularization model followed by the pterygia epithelial cell DIVAA model. The anterior segment pterygium model also showed regression histologically. This suggests that doxycycline may be successful as an adjunctive treatment for choroidal neovascularization and pterygia in humans; clinical trials would be necessary to determine if there is a benefit.

Cox, Constance A.; Amaral, Juan; Salloum, Rita; Guedez, Liliana; Reid, Ted W.; Jaworski, Cindy; John-Aryankalayil, Moly; Freedman, Ken A.; Campos, Mercedes M.; Martinez, Alfredo; Becerra, S. Patricia; Carper, Deborah A.

2010-01-01

203

The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.  

PubMed Central

The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes. Images

Wilmen, A; Pick, H; Niedenthal, R K; Sen-Gupta, M; Hegemann, J H

1994-01-01

204

The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.  

PubMed

The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes. PMID:8052535

Wilmen, A; Pick, H; Niedenthal, R K; Sen-Gupta, M; Hegemann, J H

1994-07-25

205

Genetic Analysis of Myc and Telomerase Interactions In Vivo  

PubMed Central

Myc is a transcription factor with pleiotropic effects on tumorigenesis which are likely to be mediated by its target genes. A known Myc transcriptional target is the catalytic subunit of telomerase, Tert. However, the contribution of Tert activation to Myc-induced tumorigenesis in vivo remains unknown. In this study, we addressed the role of telomerase in Myc-induced skin papillomatosis by using compound mice with a switchable Myc gene, Inv-MycERTAM mice, in combination with either telomerase deficiency (Terc?/?) or telomerase overexpression (K5-mTert) in the skin. We first demonstrated that Myc activates telomerase in the skin. With Inv-MycERTAM × Terc?/? mice, we further showed that this telomerase activation is partially required to elicit a full hyperplastic Myc-induced response. The presence of critically short telomeres in late-generation Inv-MycERTAM × Terc?/? mice further reduced the skin lesion induced by Myc. On the other hand, telomerase overexpression in the skin of K5-mTert mice augments Myc-induced hyperplasia in the absence of changes in telomere length, suggesting a direct role of telomerase in the Myc protumorigenic response. Taken together, these results highlight telomerase as a mediator of Myc-induced papillomatosis and suggest telomerase as a putative therapeutic target for Myc-dependent lesions.

Flores, Ignacio; Evan, Gerard; Blasco, Maria A.

2006-01-01

206

Multivariate Analysis of Functional Metagenomes  

PubMed Central

Metagenomics is a primary tool for the description of microbial and viral communities. The sheer magnitude of the data generated in each metagenome makes identifying key differences in the function and taxonomy between communities difficult to elucidate. Here we discuss the application of seven different data mining and statistical analyses by comparing and contrasting the metabolic functions of 212 microbial metagenomes within and between 10 environments. Not all approaches are appropriate for all questions, and researchers should decide which approach addresses their questions. This work demonstrated the use of each approach: for example, random forests provided a robust and enlightening description of both the clustering of metagenomes and the metabolic processes that were important in separating microbial communities from different environments. All analyses identified that the presence of phage genes within the microbial community was a predictor of whether the microbial community was host-associated or free-living. Several analyses identified the subtle differences that occur with environments, such as those seen in different regions of the marine environment.

Dinsdale, Elizabeth A.; Edwards, Robert A.; Bailey, Barbara A.; Tuba, Imre; Akhter, Sajia; McNair, Katelyn; Schmieder, Robert; Apkarian, Naneh; Creek, Michelle; Guan, Eric; Hernandez, Mayra; Isaacs, Katherine; Peterson, Chris; Regh, Todd; Ponomarenko, Vadim

2013-01-01

207

Biomimetic modification of metallic cardiovascular biomaterials: from function mimicking to endothelialization in vivo  

PubMed Central

Biosystem–surface interactions play an important role in various biological events and determine the ultimate functionality of implanted devices. Endothelialization or mimicking of endothelium on the surface of cardiovascular materials is a promising way to solve the problems of material-induced thrombosis and restenosis. Meanwhile, a multifunctional surface design is needed as antithrombotic properties should be considered in the period when the implants are not yet completely endothelialized. In this article, we summarize some successful approaches used in our laboratory for constructing multifunctional endothelium-like surfaces on metallic cardiovascular biomaterials through chemical modification of the surface or by the introduction of specific biological molecules to induce self-endothelialization in vivo. Some directions on future research in these areas are also presented.

Weng, Yajun; Chen, Junying; Tu, Qiufen; Li, Quanli; Maitz, Manfred F.; Huang, Nan

2012-01-01

208

In Vitro and In Vivo Evaluation of a Novel Ferrocyanide Functionalized Nanopourous Silica Decorporation Agent for Cesium (Cs) in Rats  

PubMed Central

Novel decorporation agents are being developed to protect against radiological terrorist attacks. These sorbents, known as the self-assembled monolayer on mesoporous supports (SAMMS™), are hybrid materials where differing organic moieties are grafted onto mesoporous silica (SiO2). In vitro experiments focused on the evaluation, and optimization of SAMMS for capturing radiocesium (137Cs); therefore based on these studies, a ferrocyanide copper (FC-Cu-EDA)-SAMMS was advanced for in vivo evaluation. In vivo experiments were conducted comparing the performance of the SAMMS vs. insoluble Prussian blue. Groups of jugular cannulated rats (4/treatment) were evaluated. Animals in group I were administered 137Cs chloride (~40 ?g/kg) by intravenous (iv) injection or oral gavage; Group II animals were administered pre-bound 137Cs- SAMMS or sequential 137Cs chloride + SAMMS (~61 ng/kg) by oral gavage; and Group III was orally administered 137Cs chloride (~61 ng/kg) followed by either 0.1 g of SAMMS or Prussian blue. Following dosing, the rats were maintained in metabolism cages for 72 hour and blood, urine and fecal samples were collected for 137Cs analysis (gamma counting). Rats were then humanely euthanized, and selected tissues analyzed. Orally administered 137Cs chloride was rapidly and well absorbed (~100% relative to iv dose), and the pharmacokinetics (blood, urine, feces & tissues) were very comparable to the iv dose group. For both exposures the urine and feces accounted for 20 and 3% of the dose, respectively. The prebound 137Cs-SAMMS was retained primarily within the feces (72% of the dose), with ~1.4% detected in the urine, suggesting that the 137Cs remained tightly bound to SAMMS. SAMMS & Prussian blue both effectively captured available 137Cs in the gut with feces accounting for 80–88% of the administered dose, while less than 2% was detected in the urine. This study suggests that the functionalized SAMMS outperforms Prussian blue in vitro at low pH, but demonstrates comparable in vivo sequestration efficacy at low exposure concentrations. The comparable response may be the result of the low 137Cs chloride dose and high sorbent dosage that was utilized. Future studies are planned to optimize SAMMS in vivo performance over a broader range of doses and conditions.

Timchalk, Charles; Creim, Jeffrey A; Sukwarotwat, Vichaya; Wiacek, Robert; Addleman, R Shane; Fryxell, Glen E; Yantasee, Wassana

2009-01-01

209

Ex vivo magnetofection: A novel strategy for the study of gene function in mouse organogenesis  

PubMed Central

Gene function during mouse development is often studied through the production and analysis of transgenic and knock-out models. However, these techniques are time- and resource-consuming, and require specialized equipment and expertise. We have established a new protocol for functional studies that combines organ culture of explanted fetal tissues with micro-injection and magnetically-induced transfection (“magnetofection”) of gene expression constructs. As proof-of-principle, we magnetofected cDNA constructs into genital ridge tissue as a means of gain-of-function analysis, and shRNA constructs for loss-of-function analysis. Ectopic expression of Sry induced female-to-male sex-reversal, whereas knockdown of Sox9 expression caused male-to-female sex-reversal, consistent with the known functions of these genes. Further, ectopic expression of Tmem184a, a gene of unknown function, in female genital ridges, resulted in failure of gonocytes to enter meiosis. This technique will likely be applicable to the study of gene function in a broader range of developing organs and tissues.

Svingen, Terje; Wilhelm, Dagmar; Combes, Alexander N.; Hosking, Brett; Harley, Vincent R.; Sinclair, Andrew H.; Koopman, Peter

2010-01-01

210

HIV Type 1 Infection Up-Regulates TLR2 and TLR4 Expression and Function in Vivo and in Vitro  

PubMed Central

Abstract Toll-like receptors (TLRs) play a critical role in innate immunity against pathogens. Their stimulation induces the activation of NF-?B, an important inducer of HIV-1 replication. In recent years, an increasing number of studies using several cells types from HIV-infected patients indicate that TLRs play a key role in regulating the expression of proinflammatory cytokines and viral pathogenesis. In the present study, the effect of HIV-1 stimulation of monocyte-derived macrophage (MDM) and peripheral blood mononuclear cell (PBMC) subpopulations from healthy donors on the expression and functions of TLR2 and TLR4 was examined. In addition, and to complete the in vitro study, the expression pattern of TLR2 and TLR4 in 49 HIV-1-infected patients, classified according to viral load and the use of HAART, was determined and compared with 25 healthy subjects. An increase of TLR expression and production of proinflammatory cytokines were observed in MDMs and PBMCs infected with HIV-1 in vitro and in response to TLR stimulation, compared to the mock. In addition, an association between TLR expression and up-regulation of CD80 in plasmacytoid dendritic cells (pDCs) was observed. The ex vivo analysis indicated increased expression of TLR2 and TLR4 in myeloid dendritic cells (mDCs), but only of TLR2 in monocytes obtained from HIV-1-infected patients, compared to healthy subjects. Remarkably, the expression was higher in cells from patients who do not use HAART. In monocytes, there was a positive correlation between both TLRs and viral load, but not CD4+ T cell numbers. Together, our in vitro and ex vivo results suggest that TLR expression and function can be up-regulated in response to HIV-1 infection and could affect the inflammatory response. We propose that modulation of TLRs represents a mechanism to promote HIV-1 replication or AIDS progression in HIV-1-infected patients.

Hernandez, Juan C.; Stevenson, Mario; Latz, Eicke

2012-01-01

211

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation.  

PubMed

Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior. PMID:22080836

Coffee, R Lane; Williamson, Ashley J; Adkins, Christopher M; Gray, Marisa C; Page, Terry L; Broadie, Kendal

2011-11-11

212

In vivo neuronal function of the fragile X mental retardation protein is regulated by phosphorylation  

PubMed Central

Fragile X syndrome (FXS), caused by loss of the Fragile X Mental Retardation 1 (FMR1) gene product (FMRP), is the most common heritable cause of intellectual disability and autism spectrum disorders. It has been long hypothesized that the phosphorylation of serine 500 (S500) in human FMRP controls its function as an RNA-binding translational repressor. To test this hypothesis in vivo, we employed neuronally targeted expression of three human FMR1 transgenes, including wild-type (hFMR1), dephosphomimetic (S500A-hFMR1) and phosphomimetic (S500D-hFMR1), in the Drosophila FXS disease model to investigate phosphorylation requirements. At the molecular level, dfmr1 null mutants exhibit elevated brain protein levels due to loss of translational repressor activity. This defect is rescued for an individual target protein and across the population of brain proteins by the phosphomimetic, whereas the dephosphomimetic phenocopies the null condition. At the cellular level, dfmr1 null synapse architecture exhibits increased area, branching and bouton number. The phosphomimetic fully rescues these synaptogenesis defects, whereas the dephosphomimetic provides no rescue. The presence of Futsch-positive (microtubule-associated protein 1B) supernumerary microtubule loops is elevated in dfmr1 null synapses. The human phosphomimetic restores normal Futsch loops, whereas the dephosphomimetic provides no activity. At the behavioral level, dfmr1 null mutants exhibit strongly impaired olfactory associative learning. The human phosphomimetic targeted only to the brain-learning center restores normal learning ability, whereas the dephosphomimetic provides absolutely no rescue. We conclude that human FMRP S500 phosphorylation is necessary for its in vivo function as a neuronal translational repressor and regulator of synaptic architecture, and for the manifestation of FMRP-dependent learning behavior.

Coffee, R. Lane; Williamson, Ashley J.; Adkins, Christopher M.; Gray, Marisa C.; Page, Terry L.; Broadie, Kendal

2012-01-01

213

Atypical Membrane Topology and Heteromeric Function of Drosophila Odorant Receptors In Vivo  

PubMed Central

Drosophila olfactory sensory neurons (OSNs) each express two odorant receptors (ORs): a divergent member of the OR family and the highly conserved, broadly expressed receptor OR83b. OR83b is essential for olfaction in vivo and enhances OR function in vitro, but the molecular mechanism by which it acts is unknown. Here we demonstrate that OR83b heterodimerizes with conventional ORs early in the endomembrane system in OSNs, couples these complexes to the conserved ciliary trafficking pathway, and is essential to maintain the OR/OR83b complex within the sensory cilia, where odor signal transduction occurs. The OR/OR83b complex is necessary and sufficient to promote functional reconstitution of odor-evoked signaling in sensory neurons that normally respond only to carbon dioxide. Unexpectedly, unlike all known vertebrate and nematode chemosensory receptors, we find that Drosophila ORs and OR83b adopt a novel membrane topology with their N-termini and the most conserved loops in the cytoplasm. These loops mediate direct association of ORs with OR83b. Our results reveal that OR83b is a universal and integral part of the functional OR in Drosophila. This atypical heteromeric and topological design appears to be an insect-specific solution for odor recognition, making the OR/OR83b complex an attractive target for the development of highly selective insect repellents to disrupt olfactory-mediated host-seeking behaviors of insect disease vectors.

Benton, Richard; Sachse, Silke; Michnick, Stephen W

2006-01-01

214

In vivo optogenetic tracing of functional corticocortical connections between motor forelimb areas  

PubMed Central

Interactions between distinct motor cortical areas are essential for coordinated motor behaviors. In rodents, the motor cortical forelimb areas are divided into at least two distinct areas: the rostral forelimb area (RFA) and the caudal forelimb area (CFA). The RFA is thought to be an equivalent of the premotor cortex (PM) in primates, whereas the CFA is believed to be an equivalent of the primary motor cortex. Although reciprocal connections between the RFA and the CFA have been anatomically identified in rats, it is unknown whether there are functional connections between these areas that can induce postsynaptic spikes. In this study, we used an in vivo Channelrhodopsin-2 (ChR2) photostimulation method to trace the functional connections between the mouse RFA and CFA. Simultaneous electrical recordings were utilized to detect spiking activities induced by synaptic inputs originating from photostimulated areas. This method, in combination with anatomical tracing, demonstrated that the RFA receives strong functional projections from layer 2/3 and/or layer 5a, but not from layer 5b (L5b), of the CFA. Further, the CFA receives strong projections from L5b neurons of the RFA. The onset latency of electrical responses evoked in remote areas upon photostimulation of the other areas was approximately 10 ms, which is consistent with the synaptic connectivity between these areas. Our results suggest that neuronal activities in the RFA and the CFA during movements are formed through asymmetric reciprocal connections.

Hira, Riichiro; Ohkubo, Fuki; Tanaka, Yasuhiro R.; Masamizu, Yoshito; Augustine, George J.; Kasai, Haruo; Matsuzaki, Masanori

2013-01-01

215

A tissue adhesives evaluated in vitro and in vivo analysis.  

PubMed

In this study, three kinds of two-component adhesive glues were prepared, namely, gel-dext glue made from modified gelatin and dextran, gel-HES glue made from modified gelatin and hydroxyethyl starch (HES), and chit-dext glue made from chitosan and modified dextran. Upon mixing the two-component solution together crosslinking occurred and a gel formed in several seconds, which would seal the wound tissue and stop the bleeding. The adhesive ability of those three prepared glues was evaluated in vitro and in vivo separately by measuring the bonding strength to two piece of porcine skin and the adhesive strength after sealing the skin incisions on the back of rat. Fibrin glue was used as comparing. Gel-dext glue and gel-HES glue shown higher bonding strength and adhesive strength than chit-dext glue and fibrin glue. Histology test of incision tissues given by both HE and MTC methods, the former shown that gel-dext and gel-HES glues, like fibrin glue, have only normal initial inflammation to skin tissue, which almost disappear from 9 days but chit-dext glue seams have heaver inflammation, which may last to 12 days; the later shown gel-dext and gel-HES glues similar to fibrin glue, can heal the wound fast than that of chit-dext glue. The hemostatic ability for gel-HES glue was also tested on a cut liver of rat, which depend on the gel formation speed when the two-composite solutions were mixed together. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID:20496438

Mo, Xiumei; Iwata, Hiroo; Ikada, Yoshito

2010-07-01

216

Application of Functional Analysis in Fluid Mechanics.  

National Technical Information Service (NTIS)

The work was concerned with the application of functional analysis to fluid mechanics. The specific subject dealt with is that of closed splines which were introduced by the principal investigator as the appropriate splines to solve interpolation problems...

L. G. Napolitano

1980-01-01

217

Functional analysis and treatment of coprophagia.  

PubMed

In the current investigation, functional analysis results suggested that coprophagia, the ingestion of fecal matter, was maintained by automatic reinforcement. Providing noncontingent access to alternative stimuli decreased coprophagia, and the intervention was generalized to two settings. PMID:21541128

Ing, Anna D; Roane, Henry S; Veenstra, Rebecca A

2011-01-01

218

FUNCTIONAL ANALYSIS AND TREATMENT OF COPROPHAGIA  

PubMed Central

In the current investigation, functional analysis results suggested that coprophagia, the ingestion of fecal matter, was maintained by automatic reinforcement. Providing noncontingent access to alternative stimuli decreased coprophagia, and the intervention was generalized to two settings.

Ing, Anna D; Roane, Henry S; Veenstra, Rebecca A

2011-01-01

219

In vivo enhancer analysis of human conserved non-coding sequences  

Microsoft Academic Search

Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and

Len A. Pennacchio; Nadav Ahituv; Alan M. Moses; Shyam Prabhakar; Marcelo A. Nobrega; Malak Shoukry; Simon Minovitsky; Inna Dubchak; Amy Holt; Keith D. Lewis; Ingrid Plajzer-Frick; Jennifer Akiyama; Sarah de Val; Veena Afzal; Brian L. Black; Olivier Couronne; Michael B. Eisen; Axel Visel; Edward M. Rubin

2006-01-01

220

In vivo analysis of human nucleoporin repeat domain interactions.  

PubMed

The nuclear pore complex (NPC), assembled from ?30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine-glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein-Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine-leucine-FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier. PMID:23427268

Xu, Songli; Powers, Maureen A

2013-02-20

221

In vivo analysis of human nucleoporin repeat domain interactions  

PubMed Central

The nuclear pore complex (NPC), assembled from ?30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine–glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein–Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine–leucine–FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier.

Xu, Songli; Powers, Maureen A.

2013-01-01

222

A boundary function for multicarrier multipaction analysis  

Microsoft Academic Search

This paper provides an analytical expression of a new boundary function for multipaction analysis in multicarrier operation. This boundary function is applicable for the general case where the signal amplitude of each carrier and the frequency spacing between each adjacent carrier of the multicarrier signal are different.

Jean-Christophe Angevain; Luca Salghetti Drioli; Pablo Sarasa Delgado; Cyril Mangenot

2009-01-01

223

Us3, a multifunctional protein kinase encoded by herpes simplex virus 1: how does it function in vivo?  

PubMed

: Phosphorylation is a common protein modification by which a cell or virus regulates protein activity, and subsequently cellular and viral functions. Herpesviruses commonly encode protein kinases that regulate their own replicative processes and modify host cellular machinery, by phosphorylating target proteins. Although numerous studies have revealed the multiple downstream effects of viral protein kinases and their potential molecular mechanisms, it remains unknown whether herpes viral protein kinases are involved in viral replication and pathogenicity in vivo. This review focuses on Us3 protein kinase encoded by herpes simplex virus 1 and provides a current overview of its functions in infected cells, with a special focus on their relevancy in vivo. PMID:24104928

Kawaguchi, Yasushi

2013-11-01

224

Human Retinal Pigment Epithelium Cells as Functional Models for the RPE In Vivo  

PubMed Central

Purpose. The two most commonly used in vitro models of the retinal pigment epithelium (RPE) are fetal human RPE (fhRPE) and ARPE-19 cells; however, studies of their barrier properties have produced contradictory results. To compare their utility as RPE models, their morphologic and functional characteristics were analyzed. Methods. Monolayers of both cell types were grown on permeable membrane filters. Barrier function and cellular morphology were assessed by transepithelial resistance (TER) measurements and immunohistochemistry. Protein expression was evaluated by immunoblotting and ELISA assays, and retinoid metabolism characterized by HPLC. Results. Both cultures developed tight junctions. However, only the fhRPE cells were pigmented, uniform in size and shape, expressed high levels of RPE markers, metabolized all-trans retinal, and developed high TER (>400 ?cm2). The net secretion of pigment-epithelium-derived factor (PEDF) was directed apically in both cultures, but fhRPE cells exhibited secretion rates a thousand-fold greater than in ARPE-19 cells. The net secretion of vascular endothelial growth factor (VEGF) was significantly higher in fhRPE cultures and the direction of this secretion was basolateral; while net secretion was apical in ARPE-19 cells. In fresh media, VEGF-E reduced TER in both cultures; however, in conditioned media fhRPE cells did not respond to VEGF-E administration, but retreatment of the conditioned media with anti-PEDF antibodies allowed fhRPE cells to fully respond to VEGF-E. Conclusions. Properties of fhRPE cells align with a functionally normal RPE in vivo, while ARPE-19 cells resemble a pathologic or aged RPE. These results suggest a utility for both cell types in understanding distinct, particular aspects of RPE function.

Dahrouj, Mohammad; Tang, Peter H.; Liu, Yueying; Sambamurti, Kumar; Marmorstein, Alan D.; Crosson, Craig E.

2011-01-01

225

Conditional gene deletion reveals functional redundancy of GABAB receptors in peripheral nociceptors in vivo  

PubMed Central

Background ?-aminobutyric acid (GABA) is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear. Results In order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1) subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1)-/- mice). Lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1)-/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1)-/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1)-/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1) expression in nociceptors. Conclusion This study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.

2009-01-01

226

Recovery of macular pigment spectrum in vivo using hyperspectral image analysis  

NASA Astrophysics Data System (ADS)

We investigated the feasibility of a novel method for hyperspectral mapping of macular pigment (MP) in vivo. Six healthy subjects were recruited for noninvasive imaging using a snapshot hyperspectral system. The three-dimensional full spatial-spectral data cube was analyzed using non-negative matrix factorization (NMF), wherein the data was decomposed to give spectral signatures and spatial distribution, in search for the MP absorbance spectrum. The NMF was initialized with the in vitro MP spectrum and rank 4 spectral signature decomposition was used to recover the MP spectrum and optical density in vivo. The recovered MP spectra showed two peaks in the blue spectrum, characteristic of MP, giving a detailed in vivo demonstration of these absorbance peaks. The peak MP optical densities ranged from 0.08 to 0.22 (mean 0.15+/-0.05) and became spatially negligible at diameters 1100 to 1760 ?m (4 to 6 deg) in the normal subjects. This objective method was able to exploit prior knowledge (the in vitro MP spectrum) in order to extract an accurate in vivo spectral analysis and full MP spatial profile, while separating the MP spectra from other ocular absorbers. Snapshot hyperspectral imaging in combination with advanced mathematical analysis provides a simple cost-effective approach for MP mapping in vivo.

Fawzi, Amani A.; Lee, Noah; Acton, Jennifer H.; Laine, Andrew F.; Smith, R. Theodore

2011-10-01

227

Functional significance of glutamate-cysteine ligase modifier for erythrocyte survival in vitro and in vivo.  

PubMed

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm(-/-)) mice. Erythrocytes from gclm(-/-) mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm(-/-) erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07?mg/g b.w.) to induce oxidative stress. Gclm(-/-) mice showed dramatically increased hemolysis compared with gclm(+/+) controls. In addition, PHZ-treated gclm(-/-) mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm(+/+) mice within 24?h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm(-/-) mice and none in gclm(+/+) controls. In fact, 24?h after treatment, kidney function began to diminish in gclm(-/-) mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm(+/+) mice survived, 90% of PHZ-treated gclm(-/-) mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm(-/-) erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm(+/+) erythrocytes, an effect at least partially due to increased cytosolic Ca(2+) concentration. Under resting conditions, gclm(-/-) mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress. PMID:23787995

Föller, M; Harris, I S; Elia, A; John, R; Lang, F; Kavanagh, T J; Mak, T W

2013-06-21

228

Characterization of in vivo functions of Nicotiana benthamiana RabE1.  

PubMed

We characterized the gene expression, subcellular localization, and in vivo functions of a Nicotiana benthamiana small GTPase belonging to the RabE family, designated NbRabE1. The NbRabE1 promoter drove strong ?-glucuronidase reporter expression in young tissues containing actively dividing cells and in stomata guard cells. GFP fusion proteins of NbRabE1 and its dominant-negative and constitutively active mutants were all localized to the Golgi apparatus and the plasma membrane but showed different affinities for membrane attachment. Virus-induced gene silencing of NbRabE1 resulted in pleiotropic phenotypes, including growth arrest, premature senescence, and abnormal leaf development. At the cellular level, the leaves in which NbRabE1 was silenced contained abnormal stomata that lacked pores or contained incomplete ventral walls, suggesting that NbRabE1 deficiency leads to defective guard cell cytokinesis. Ectopic expression of the dominant-negative mutant of NbRabE1 in Arabidopsis thaliana resulted in retardation of shoot and root growth accompanied by defective root hair formation. These developmental defects are discussed in conjunction with proposed functions of RabE GTPases in polarized secretory vesicle trafficking. PMID:23001196

Ahn, Chang Sook; Han, Jeong-A; Pai, Hyun-Sook

2012-09-22

229

Humanized large-scale expanded endothelial colony-forming cells function in vitro and in vivo  

PubMed Central

Endothelial progenitor cells are critically involved in essential biologic processes, such as vascular homeostasis, regeneration, and tumor angiogenesis. Endothelial colony–forming cells (ECFCs) are endothelial progenitor cells with robust proliferative potential. Their profound vessel-forming capacity makes them a promising tool for innovative experimental, diagnostic, and therapeutic strategies. Efficient and safe methods for their isolation and expansion are presently lacking. Based on the previously established efficacy of animal serum–free large-scale clinical-grade propagation of mesenchymal stromal cells, we hypothesized that endothelial lineage cells may also be propagated efficiently following a comparable strategy. Here we demonstrate that human ECFCs can be recovered directly from unmanipulated whole blood. A novel large-scale animal protein-free humanized expansion strategy preserves the progenitor hierarchy with sustained proliferation potential of more than 30 population doublings. By applying large-scale propagated ECFCs in various test systems, we observed vascular networks in vitro and perfused vessels in vivo. After large-scale expansion and cryopreservation phenotype, function, proliferation, and genomic stability were maintained. For the first time, proliferative, functional, and storable ECFCs propagated under humanized conditions can be explored in terms of their therapeutic applicability and risk profile.

Reinisch, Andreas; Hofmann, Nicole A.; Obenauf, Anna C.; Kashofer, Karl; Rohde, Eva; Schallmoser, Katharina; Flicker, Karin; Lanzer, Gerhard; Linkesch, Werner; Speicher, Michael R.

2009-01-01

230

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation  

PubMed Central

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

Psaila, Bethan; Bussel, James B.; Linden, Matthew D.; Babula, Bracken; Li, Youfu; Barnard, Marc R.; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L.

2012-01-01

231

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation.  

PubMed

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased. PMID:22294727

Psaila, Bethan; Bussel, James B; Linden, Matthew D; Babula, Bracken; Li, Youfu; Barnard, Marc R; Tate, Chinara; Mathur, Kanika; Frelinger, Andrew L; Michelson, Alan D

2012-01-31

232

Effects of altered ventilatory patterns of rabbit pulmonary endothelial angiotensin converting enzyme function, in vivo  

SciTech Connect

Because alveolar pressure can influence pulmonary blood flow, volume and surface area, the authors have studied the effects of airway pressure on endothelial angiotensin converting enzyme (ACE) function in rabbit lungs in vivo, utilizing indicator dilution techniques with /sup 3/H-Benzoyl-Phe-Ala-Pro (BPAP) as substate. Static inclation of the lungs to a pressure of 0 or 5 mmHg did not change percent transpulmonary metabolism and Amax/Km ratio in comparison to control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 mmHg, percent metabolism of /sup 3/H-BPAP remained unaltered but Amax/Km decreased over 40% from control. This decrease was in close relation to the reduction in pulmonary blood flow. Addition of 5 cm H/sub 2/O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of /sup 3/H-BPAP. These results suggest that the detected alterations in ACE kinetics were more likely due to hemodynamic changes than enzyme dysfunction. The authors propose that high static alveolar pressures as well as PEEP did not affect angiotensin converting enzyme function, but reduced the fraction of perfused microvessels reflected in changes in Amax/Km ratios.

Toivonen, H.J.; Catravas, J.D.

1986-03-01

233

Twins, quadruplexes, and more: functional aspects of native and engineered RNA self-assembly in vivo  

PubMed Central

The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA–RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self–self) or heteromeric (self–nonself) RNA–RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA–RNA self-assembly.

Lease, Richard A.; Arluison, Veronique; Lavelle, Christophe

2013-01-01

234

Monitoring of In Vivo Function of Superparamagnetic Iron Oxide Labelled Murine Dendritic Cells during Anti-Tumour Vaccination  

PubMed Central

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4+ T cells but, most importantly, they primed cytotoxic CD8+ T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer.

Tavare, Richard; Sagoo, Pervinder; Varama, Gopal; Tanriver, Yakup; Warely, Alice; Diebold, Sandra S.; Southworth, Richard; Schaeffter, Tobias; Lechler, Robert I.; Razavi, Reza; Lombardi, Giovanna; Mullen, Gregory E. D.

2011-01-01

235

Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo.  

PubMed

Telomerase is a ribonucleoprotein complex that extends the 3' ends of linear chromosomes. The specialized telomerase reverse transcriptase requires a multidomain RNA (telomerase RNA, TER), which includes an integral RNA template and functionally important template-adjacent pseudoknot. The structure of the human TER pseudoknot revealed that the loops interact with the stems to form a triple helix shown to be important for activity in vitro. A similar triple helix has been predicted to form in diverse fungi TER pseudoknots. The solution NMR structure of the Kluyveromyces lactis pseudoknot, presented here, reveals that it contains a long pyrimidine motif triple helix with unexpected features that include three individual bulge nucleotides and a C(+)•G-C triple adjacent to a stem 2-loop 2 junction. Despite significant differences in sequence and base triples, the 3D shape of the human and K. lactis TER pseudoknots are remarkably similar. Analysis of the effects of nucleotide substitutions on cell growth and telomere lengths provides evidence that this conserved structure forms in endogenously assembled telomerase and is essential for telomerase function in vivo. PMID:23776224

Cash, Darian D; Cohen-Zontag, Osnat; Kim, Nak-Kyoon; Shefer, Kinneret; Brown, Yogev; Ulyanov, Nikolai B; Tzfati, Yehuda; Feigon, Juli

2013-06-17

236

Cardiac magnetic resonance, transthoracic and transoesophageal echocardiography: a comparison of in vivo assessment of ventricular function in rats.  

PubMed

In vivo assessment of ventricular function in rodents has largely been restricted to transthoracic echocardiography (TTE). However 1.5?T cardiac magnetic resonance (CMR) and transoesophageal echocardiography (TOE) have emerged as possible alternatives. Yet, to date, no study has systematically assessed these three imaging modalities in determining ejection fraction (EF) in rats. Twenty rats underwent imaging four weeks after surgically-induced myocardial infarction. CMR was performed on a 1.5?T scanner, TTE was conducted using a 9.2?MHz transducer and TOE was performed with a 10?MHz intracardiac echo catheter. Correlation between the three techniques for EF determination and analysis reproducibility was assessed. Moderate-strong correlation was observed between the three modalities; the greatest between CMR and TOE (intraclass correlation coefficient (ICC)?=?0.89), followed by TOE and TTE (ICC?=?0.70) and CMR and TTE (ICC?=?0.63). Intra- and inter-observer variations were excellent with CMR (ICC?=?0.99 and 0.98 respectively), very good with TTE (0.90 and 0.89) and TOE (0.87 and 0.84). Each modality is a viable option for evaluating ventricular function in rats, however the high image quality and excellent reproducibility of CMR offers distinct advantages even at 1.5?T with conventional coils and software. PMID:23836849

Richardson, Jd; Bertaso, Ag; Frost, L; Psaltis, Pj; Carbone, A; Koschade, B; Wong, Dt; Nelson, Aj; Paton, S; Williams, K; Azarisman, S; Worthley, Mi; Teo, Ks; Gronthos, S; Zannettino, Acw; Worthley, Sg

2013-07-08

237

Analysis of adhesive behaviour of human skin in vivo by an indentation test  

Microsoft Academic Search

Indentation testing is a widely used technique to quantify the elastoplastic properties of materials. However, it also allows the analysis of the adhesive force between a substrate and an indenter. In this paper, in order to contribute to the understanding of the human skin behaviour, an elastic adhesive theory has been applied to a steel\\/skin in vivo contact. The human

C. Pailler-Mattéi; H. Zahouani

2006-01-01

238

In vivo activation analysis of spinal calcium using 25 keV neutrons  

SciTech Connect

The applicability of 25 keV neutrons to the in vivo activation analysis of spinal calcium is examined both theoretically and experimentally. It is shown that the use of this energy results in an increase in sensitivity over higher energy neutrons so that patient dose may be significantly reduced.

Cousins, T.; Kennett, T.J.; Prestwich, W.V.; Webber, C.E.

1980-11-01

239

ANALYSIS OF IN VITRO AND IN VIVO DNA STRAND BREAKS INDUCED BY TRIHALOMETHANES (THMS)  

EPA Science Inventory

Analysis of In Vitro and In Vivo DNA Strand Breaks Induced by Trihalomethanes (TRMs) The THMs are the most widely distributed and the most concentrated of the cWorine disinfection by-products (D BPs) found in finished drinking water. All of the THMs, cWoroform (CHCI3), br...

240

Computer aided design of a polarised source for in vivo X-ray flourescence analysis  

Microsoft Academic Search

A system has been developed which uses Monte Carlo computer simulations to aid the design and optimisatioin of a polarised source for in vivo X-ray fluorescence (XRF) analysis of heavy metals. The system is based on a version of the Monte Carlo code EGS4 which includes polarised photon interactions, running on a personal computer. The code was used to construct

D. G. Lewis; A. Kilic; C. A. Ogg

1998-01-01

241

Stress analysis of carotid plaque rupture based on in vivo high resolution MRI  

Microsoft Academic Search

Atheromatous carotid plaque rupture is responsible for the majority of ischaemic strokes in the developed world. Plaque rupture has been associated with plaque morphology, plaque components’ properties, inflammation and local stress concentration. High resolution multi-spectral magnetic resonance imaging (MRI) has allowed the plaque components to be visualized in vivo. This study combined the recent advances in finite element analysis (FEA)

Zhi-Yong Li; Simon Howarth; Rikin A. Trivedi; Jean M. U-King-Im; Martin J. Graves; Andrew Brown; Liqun Wang; Jonathan H. Gillard

2006-01-01

242

In VivoFunctional Imaging of Intrinsic Scattering Changes in the Human Retina with High-speed Ultrahigh Resolution OCT  

PubMed Central

Non-invasive methods of probing retinal function are of interest for the early detection of retinal disease. While retinal function is traditionally directly measured with the electroretinogram (ERG), recently functional optical imaging of the retina has been demonstrated. In this manuscript, stimulus-induced, intrinsic optical scattering changes in the human retina are measured in vivo with high-speed, ultrahigh resolution optical coherence tomography (OCT) operating at 50,000 axial scans per second and ?3.3 micron axial resolution. A stimulus and measurement protocol that enables measurement of functional OCT retinal signals is described. OCT signal changes in the photoreceptors are demonstrated. Two distinct responses having different temporal and spatial properties are reported. These results are discussed in the context of optical intrinsic signals measured previously in the retina by fundus imaging and scanning laser ophthalmoscopy. Finally, challenges associated with in vivo functional retinal imaging in human subjects are discussed.

Srinivasan, V. J.; Chen, Y.; Duker, J. S.; Fujimoto, J. G.

2009-01-01

243

Transcriptome Analysis in Chicken Cecal Epithelia upon Infection by Eimeria tenella In Vivo  

PubMed Central

Coccidiosis, caused by various Eimeria species, is a major parasitic disease in chickens. However, our understanding on how chickens respond to coccidian infection is highly limited at both molecular and cellular levels. The present study employed the Affymetrix chicken genome array and performed transcriptome analysis on chicken cecal epithelia in response to infection for 4.5 days in vivo by the cecal-specific species E. tenella. By Significance Analysis of Microarrays (SAM), we have identified 7,099 probe sets with q-values at <0.05, in which 4,033 and 3,066 genes were found to be up- or down-regulated in response to parasite infection. The reliability of the microarray data were validated by real-time qRT-PCR of 20 genes with varied fold changes in expression (i.e., correlation coefficient between microarray and qRT-PCR datasets: R2?=?0.8773, p<0.0001). Gene ontology analysis, KEGG pathway mapping and manual annotations of regulated genes indicated that up-regulated genes were mainly involved in immunity/defense, responses to various stimuli, apoptosis/cell death and differentiation, signal transduction and extracellular matrix (ECM), whereas down-regulated genes were mainly encoding general metabolic enzymes, membrane components, and some transporters. Chickens mustered complex cecal eipthelia molecular and immunological responses in response to E. tenella infection, which included pathways involved in cytokine production and interactions, natural killer cell mediated cytotoxicity, and intestinal IgA production. In response to the pathogenesis and damage caused by infection, chicken cecal epithelia reduced general metabolism, DNA replication and repair, protein degradation, and mitochondrial functions.

Guo, Aijiang; Cai, Jianping; Gong, Wei; Yan, Hongbin; Luo, Xuenong; Tian, Guangfu; Zhang, Shaohua; Zhang, Haili; Zhu, Guan; Cai, Xuepeng

2013-01-01

244

A dynamic analysis of a functional brace for anterior cruciate ligament insufficiency  

Microsoft Academic Search

A dynamic, in vivo, functional analysis of braces de signed for ACL insufficiency has never been reported. In this study, 14 athletes who had arthroscopically proven absent ACLs were evaluated in the Biomechan ics Laboratory at the Centinela Hospital Medical Center. None of the ligaments were repaired or reconstructed. Footswitch, high speed photography, and force place data were recorded while

Frank F. Cook; James E. Tibone; Fredrick C. Redfern

1989-01-01

245

The past, present, and future of x-ray technology for in vivo imaging of function and form  

SciTech Connect

Scientists and clinicians have a keen interest in studying not just the structure of physiological systems, but their motion also, or more generally their form and function. This paper focuses on the technologies that underpin in vivo measurements of form and function of the human body for both research and medical treatment. A concise literature review of x-ray imaging, ultrasonography, magnetic resonance imaging, radionuclide imaging, laser Doppler velocimetry, and particle image velocimetry is presented. Additionally, a more detailed review of in vivo x-ray imaging is presented. Finally, two techniques, which the authors believe are representative of the present and future of in vivo x-ray imaging techniques, are presented.

Fouras, A.; Dubsky, S.; Hourigan, K. [Division of Biological Engineering, Monash University, Clayton, Victoria 3800 (Australia) and Fluids Laboratory for Aeronautical and Industrial Research, Monash University, Clayton, Victoria 3800 (Australia); Kitchen, M. J. [School of Physics, Monash University, Clayton, Victoria 3800 (Australia); Lewis, R. A. [Monash Center for Synchrotron Science, Monash University, Clayton, Victoria 3800 (Australia); Hooper, S. B. [Department of Physiology, Monash University, Clayton, Victoria 3800 (Australia)

2009-05-15

246

Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions  

PubMed Central

Carbon sequestration, infrastructure rehabilitation, brownfields clean-up, hazardous waste disposal, water resources protection and global warming—these twenty-first century challenges can neither be solved by the high-energy consumptive practices that hallmark industry today, nor by minor tweaking or optimization of these processes. A more radical, holistic approach is required to develop the sustainable solutions society needs. Most of the above challenges occur within, are supported on, are enabled by or grown from soil. Soil, contrary to conventional civil engineering thought, is a living system host to multiple simultaneous processes. It is proposed herein that ‘soil engineering in vivo’, wherein the natural capacity of soil as a living ecosystem is used to provide multiple solutions simultaneously, may provide new, innovative, sustainable solutions to some of these great challenges of the twenty-first century. This requires a multi-disciplinary perspective that embraces the science of biology, chemistry and physics and applies this knowledge to provide multi-functional civil and environmental engineering designs for the soil environment. For example, can native soil bacterial species moderate the carbonate cycle in soils to simultaneously solidify liquefiable soil, immobilize reactive heavy metals and sequester carbon—effectively providing civil engineering functionality while clarifying the ground water and removing carbon from the atmosphere? Exploration of these ideas has begun in earnest in recent years. This paper explores the potential, challenges and opportunities of this new field, and highlights one biogeochemical function of soil that has shown promise and is developing rapidly as a new technology. The example is used to propose a generalized approach in which the potential of this new field can be fully realized.

DeJong, Jason T.; Soga, Kenichi; Banwart, Steven A.; Whalley, W. Richard; Ginn, Timothy R.; Nelson, Douglas C.; Mortensen, Brina M.; Martinez, Brian C.; Barkouki, Tammer

2011-01-01

247

Transdifferentiation of Fast Skeletal Muscle Into Functional Endothelium in Vivo by Transcription Factor Etv2  

PubMed Central

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Gomez, Gustavo A.; Lindgren, Anne G.; Huang, Haigen; Yang, Hanshuo; Yao, Shaohua; Martin, Benjamin L.; Kimelman, David; Lin, Shuo

2013-01-01

248

Soil engineering in vivo: harnessing natural biogeochemical systems for sustainable, multi-functional engineering solutions.  

PubMed

Carbon sequestration, infrastructure rehabilitation, brownfields clean-up, hazardous waste disposal, water resources protection and global warming-these twenty-first century challenges can neither be solved by the high-energy consumptive practices that hallmark industry today, nor by minor tweaking or optimization of these processes. A more radical, holistic approach is required to develop the sustainable solutions society needs. Most of the above challenges occur within, are supported on, are enabled by or grown from soil. Soil, contrary to conventional civil engineering thought, is a living system host to multiple simultaneous processes. It is proposed herein that 'soil engineering in vivo', wherein the natural capacity of soil as a living ecosystem is used to provide multiple solutions simultaneously, may provide new, innovative, sustainable solutions to some of these great challenges of the twenty-first century. This requires a multi-disciplinary perspective that embraces the science of biology, chemistry and physics and applies this knowledge to provide multi-functional civil and environmental engineering designs for the soil environment. For example, can native soil bacterial species moderate the carbonate cycle in soils to simultaneously solidify liquefiable soil, immobilize reactive heavy metals and sequester carbon-effectively providing civil engineering functionality while clarifying the ground water and removing carbon from the atmosphere? Exploration of these ideas has begun in earnest in recent years. This paper explores the potential, challenges and opportunities of this new field, and highlights one biogeochemical function of soil that has shown promise and is developing rapidly as a new technology. The example is used to propose a generalized approach in which the potential of this new field can be fully realized. PMID:20829246

DeJong, Jason T; Soga, Kenichi; Banwart, Steven A; Whalley, W Richard; Ginn, Timothy R; Nelson, Douglas C; Mortensen, Brina M; Martinez, Brian C; Barkouki, Tammer

2010-09-09

249

Protein Polymer MRI Contrast Agents: Longitudinal Analysis of Biomaterials in Vivo  

PubMed Central

Despite recent advances in tissue engineering to regenerate biological function by combining cells with material supports, development is hindered by inadequate techniques for characterizing biomaterials in vivo. Magnetic Resonance Imaging (MRI) is a tomographic technique with high temporal and spatial resolution and represents an excellent imaging modality for longitudinal non-invasive assessment of biomaterials in vivo. To distinguish biomaterials from surrounding tissues for MR imaging, protein polymer contrast agents (PPCAs) were developed and incorporated into hydrogels. In vitro and in vivo images of protein polymer hydrogels, with and without covalently incorporated PPCAs, were acquired by MRI. T1 values of the labeled gels were consistently lower when PPCAs were included. As a result, the PPCA hydrogels facilitated fate tracking, quantification of degradation, and detection of immune response in vivo. For the duration of the in vivo study, the PPCA-containing hydrogels could be distinguished from adjacent tissues and from the foreign body response surrounding the gels. The hydrogels containing PPCA have a contrast-to-noise ratio two-fold greater than hydrogels without PPCA. In the absence of the PPCA, hydrogels cannot be distinguished by the end of the gel lifetime.

Karfeld-Sulzer, Lindsay S.; Waters, Emily A.; Kohlmeir, Ellen K.; Kissler, Hermann; Zhang, Xiaomin; Kaufman, Dixon B.; Barron, Annelise E.; Meade, Thomas J.

2010-01-01

250

In vivo analysis of synaptonemal complex formation during yeast meiosis.  

PubMed Central

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.

White, Eric J; Cowan, Carrie; Cande, W Zacheus; Kaback, David B

2004-01-01

251

An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function  

Microsoft Academic Search

correlates with a reduction in both the basal and assem- bly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regu- lated by its GAP activity. Based on these and other obser- vations, we propose a two-step model for

Radhakrishnan Narayanan; Marilyn Leonard; Byeong Doo Song; Sandra L. Schmid; Mani Ramaswami

2005-01-01

252

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas  

PubMed Central

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in Cas substrate domain. These tyrosine phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas-Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-lengh Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.

Roselli, Severine; Wallez, Yann; Wang, Lei; Vervoort, Virginie; Pasquale, Elena B

2010-01-01

253

In Vivo Characterization of Traumatic Brain Injury Neuropathology with Structural and Functional Neuroimaging  

PubMed Central

Quantitative neuroimaging is increasingly used to study the effects of traumatic brain injury (TBI) on brain structure and function. This paper reviews quantitative structural and functional neuroimaging studies of patients with TBI, with an emphasis on the effects of diffuse axonal injury (DAI), the primary neuropathology in TBI. Quantitative structural neuroimaging has evolved from simple planometric measurements through targeted region-of-interest analyses to whole-brain analysis of quantified tissue compartments. Recent studies converge to indicate widespread volume loss of both gray and white matter in patients with moderate-to-severe TBI. These changes can be documented even when patients with focal lesions are excluded. Broadly speaking, performance on standard neuropsychological tests of speeded information processing are related to these changes, but demonstration of specific brain-behavior relationships requires more refined experimental behavioral measures. The functional consequences of these structural changes can be imaged with activation functional neuroimaging. Although this line of research is at an early stage, results indicate that TBI causes a more widely dispersed activation in frontal and posterior cortices. Further progress in analysis of the consequences of TBI on neural structure and function will require control of variability in neuropathology and behavior.

LEVINE, BRIAN; FUJIWARA, ESTHER; O'CONNOR, CHARLENE; RICHARD, NADINE; KOVACEVIC, NATASA; MANDIC, MARINA; RESTAGNO, ADRIANA; EASDON, CRAIG; ROBERTSON, IAN H.; GRAHAM, SIMON J.; CHEUNG, GORDON; GAO, FUQIANG; SCHWARTZ, MICHAEL L.; BLACK, SANDRA E.

2007-01-01

254

Rank estimation and the multivariate analysis of in vivo fast-scan cyclic voltammetric data  

PubMed Central

Principal component regression has been used in the past to separate current contributions from different neuromodulators measured with in vivo fast-scan cyclic voltammetry. Traditionally, a percent cumulative variance approach has been used to determine the rank of the training set voltammetric matrix during model development, however this approach suffers from several disadvantages including the use of arbitrary percentages and the requirement of extreme precision of training sets. Here we propose that Malinowski’s F-test, a method based on a statistical analysis of the variance contained within the training set, can be used to improve factor selection for the analysis of in vivo fast-scan cyclic voltammetric data. These two methods of rank estimation were compared at all steps in the calibration protocol including the number of principal components retained, overall noise levels, model validation as determined using a residual analysis procedure, and predicted concentration information. By analyzing 119 training sets from two different laboratories amassed over several years, we were able to gain insight into the heterogeneity of in vivo fast-scan cyclic voltammetric data and study how differences in factor selection propagate throughout the entire principal component regression analysis procedure. Visualizing cyclic voltammetric representations of the data contained in the retained and discarded principal components showed that using Malinowski’s F-test for rank estimation of in vivo training sets allowed for noise to be more accurately removed. Malinowski’s F-test also improved the robustness of our criterion for judging multivariate model validity, even though signal-to-noise ratios of the data varied. In addition, pH change was the majority noise carrier of in vivo training sets while dopamine prediction was more sensitive to noise.

Keithley, Richard B.; Carelli, Regina M.; Wightman, R. Mark

2010-01-01

255

3.5 Years of Insulin Therapy With Insulin Glargine Improves In Vivo Endothelial Function in Type 2 Diabetes  

Microsoft Academic Search

Objective—To determine long-term effects of insulin glargine on vascular function in patients with type 2 diabetes. Methods and Results—A total of 49 in vivo endothelial function tests, intrabrachial artery infusions of endothelium- dependent (acetylcholine (ACh)) and endothelium-independent (sodium nitroprusside (SNP)) vasoactive agents, were performed in 11 patients with type 2 diabetes (age: 592 years; BMI: 29.70.9 kg\\/m2; fasting plasma glucose:

Satu Vehkavaara; Hannele Yki-Jarvinen

2010-01-01

256

Structure-function relationships in feedback regulation of energy fluxes in vivo in health and disease: mitochondrial interactosome.  

PubMed

The aim of this review is to analyze the results of experimental research of mechanisms of regulation of mitochondrial respiration in cardiac and skeletal muscle cells in vivo obtained by using the permeabilized cell technique. Such an analysis in the framework of Molecular Systems Bioenergetics shows that the mechanisms of regulation of energy fluxes depend on the structural organization of the cells and interaction of mitochondria with cytoskeletal elements. Two types of cells of cardiac phenotype with very different structures were analyzed: adult cardiomyocytes and continuously dividing cancerous HL-1 cells. In cardiomyocytes mitochondria are arranged very regularly, and show rapid configuration changes of inner membrane but no fusion or fission, diffusion of ADP and ATP is restricted mostly at the level of mitochondrial outer membrane due to an interaction of heterodimeric tubulin with voltage dependent anion channel, VDAC. VDAC with associated tubulin forms a supercomplex, Mitochondrial Interactosome, with mitochondrial creatine kinase, MtCK, which is structurally and functionally coupled to ATP synthasome. Due to selectively limited permeability of VDAC for adenine nucleotides, mitochondrial respiration rate depends almost linearly upon the changes of cytoplasmic ADP concentration in their physiological range. Functional coupling of MtCK with ATP synthasome amplifies this signal by recycling adenine nucleotides in mitochondria coupled to effective phosphocreatine synthesis. In cancerous HL-1 cells this complex is significantly modified: tubulin is replaced by hexokinase and MtCK is lacking, resulting in direct utilization of mitochondrial ATP for glycolytic lactate production and in this way contributing in the mechanism of the Warburg effect. Systemic analysis of changes in the integrated system of energy metabolism is also helpful for better understanding of pathogenesis of many other diseases. PMID:20096261

Saks, Valdur; Guzun, Rita; Timohhina, Natalja; Tepp, Kersti; Varikmaa, Minna; Monge, Claire; Beraud, Nathalie; Kaambre, Tuuli; Kuznetsov, Andrey; Kadaja, Lumme; Eimre, Margus; Seppet, Enn

2010-01-21

257

The functional response of upstream DNA to dynamic supercoiling in vivo.  

PubMed

Because RNA polymerase is a powerful motor, transmission of transcription-generated forces might directly alter DNA structure, chromatin or gene activity in mammalian cells. Here we show that transcription-generated supercoils streaming dynamically from active promoters have considerable consequences for DNA structure and function in cells. Using a tamoxifen-activatable Cre recombinase to excise a test segment of chromatin positioned between divergently transcribed metallothionein-IIa promoters, we found the degree of dynamic supercoiling to increase as transcription intensified, and it was very sensitive to the specific arrangement of promoters and cis elements. Using psoralen as an in vivo probe confirmed that, during transcription, sufficient supercoiling is produced to enable transitions to conformations other than B-DNA in elements such as the human MYC far upstream element (FUSE), which in turn recruit structure-sensitive regulatory proteins, such as FUSE Binding Protein (FBP) and FBP-Interacting Repressor (FIR). These results indicate that mechanical stresses, constrained by architectural features of DNA and chromatin, may broadly contribute to gene regulation. PMID:18193062

Kouzine, Fedor; Sanford, Suzanne; Elisha-Feil, Zichrini; Levens, David

2008-01-13

258

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.  

PubMed

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates. PMID:23204504

Rudolf, Mareike; Machettira, Anu B; Groß, Lucia E; Weber, Katrin L; Bolte, Kathrin; Bionda, Tihana; Sommer, Maik S; Maier, Uwe G; Weber, Andreas P M; Schleiff, Enrico; Tripp, Joanna

2012-11-30

259

In vivo and in vitro effects of 1,1-dimethylhydrazine on selected immune functions.  

PubMed

The in vivo phase of the experiments reported here include the evaluation of immune function after short-or long-term treatment of mice with 1,1-dimethylhydrazine (UDMH). Long-term exposure (3 injections/week for 14 weeks) resulted in increased numbers of Jerne plaque-forming cells, a trend toward decreased induction of suppressor cell activity by concanavalin A (Con A), and no effects on mitogen-induced lymphocyte blast transformation (LBT), compared to saline-treated control mice. These effects were greatest at doses of 10 or 50 mg/kg, while higher doses had less of an effect. In vitro experiments were performed by adding UDMH to normal murine splenocytes in the LBT assay and con A-induced suppressor cell assay. The UDMH induced a significant enhanced response to lipopolysaccharide (LPS) at 10 and 50 micrograms/ml, and a suppressed response to both Con A and LPS at higher concentrations. The UDMH also caused a decrease in suppressor cell activity at 25 micrograms/ml. Selective abrogation of suppressor activity or alteration of the suppressor cell-helper ratio were suggested as possible mechanisms for the enhancement effect associated with UDMH. PMID:6211418

Tarr, M J; Olsen, R G; Jacobs, D L

1982-04-01

260

3'-untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation.  

PubMed Central

Recent findings have indicated that the 3'-untranslated region (3'-UTR) of the mRNA encoding the beta-catalytic subunit of the mitochondrial H(+)-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255-5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849-855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3'-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3'-UTR of the mRNAs encoding the alpha- and beta-subunits of the rat H(+)-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3'-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3'-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.

Di Liegro, C M; Bellafiore, M; Izquierdo, J M; Rantanen, A; Cuezva, J M

2000-01-01

261

A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo.  

PubMed

The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other. PMID:10692552

Trézéguet, V; Le Saux, A; David, C; Gourdet, C; Fiore, C; Dianoux, A; Brandolin, G; Lauquin, G J

2000-02-24

262

Identification and functional characterization in vivo of a novel splice variant of LDLR in rhesus macaques.  

PubMed

In the course of developing a low-density lipoprotein receptor (LDLR) gene therapy treatment for homozygous familial hypercholesterolemia (HoFH), we planned to examine the efficacy in a nonhuman primate model, the rhesus macaque heterozygous for an LDL receptor mutation fed a high-fat diet. Unexpectedly, our initial cDNA sequencing studies led to the identification of a heretofore unidentified splicing isoform of the rhesus LDLR gene. Compared with the publicly available GenBank reference sequence of rhesus LDLR, the novel isoform contains a 21 bp in frame insertion. This sequence coincides with part of exon 5 and creates a site for the restriction enzyme MscI. Using this site as a marker for the 21 bp in-frame insertion, we conducted a restriction enzyme screen to examine for the prevalence of the novel isoform in rhesus liver tissue cDNA and its homolog in human liver tissue cDNA. We found that the novel isoform is the predominant LDLR cDNA found in rhesus liver and the sole LDLR cDNA found in human liver. Finally, we compared the in vivo functionality of the novel and previously identified rhesus LDLR splicing isoforms in a mouse model of HoFH. PMID:21628398

Kassim, Sadik H; Vandenberghe, Luk H; Hovhannisyan, Ruben; Wilson, James M; Rader, Daniel J

2011-05-31

263

[In vivo studies of the main functional systems in the heteronemertean pilidium larva].  

PubMed

There is performed in vivo morphological study of the White Sea heteronemerteans belonging to the type of pilidium pyramidale (conussoidale). Based on the layer-by-layer microshooting with subsequent computer processing, development of the pilidium digestive, nervous, and muscle systems is described from the stage following at once the gastrula to the premetamorphose larva. Peculiarities of structural organization of the main functional systems are revealed depending on the larva size and the stage of formation of imaginal discs. It is first shown that even in the not completely formed pilidium, neurons are located not only in integuments and wall of the digestive tract, but also in the depth of cupola along the central muscle retractor. Their processes are distributed between the main body parts and organs by seeming to perform connections of the apical organ and central muscle retractor with the digestive tract, blades, and the nerve plexus of the cupola wall. In the digestive tract between pharynx and stomach in the formed pilidium, the sphincter is first revealed. It has been shown that in the course of larva development, the non-orderly arranged and poorly developed muscle fibers gradually form in the blade the fan-like, whereas in the cupola wall, the net-like structure. PMID:20799611

Za?tseva, O V; Fliachinskaia, L P

264

In vivo functional and myeloarchitectonic mapping of human primary auditory areas  

PubMed Central

In contrast to vision, where retinotopic mapping alone can define areal borders, primary auditory areas such as A1 are best delineated by combining in vivo tonotopic mapping with post mortem cyto- or myelo-architectonics from the same individual. We combined high-resolution (800 ?m) quantitative T1 mapping with phase-encoded tonotopic methods to map primary auditory areas (A1 and R) within the ‘auditory core’ of human volunteers. We first quantitatively characterize the highly myelinated auditory core in terms of shape, area, cortical depth profile, and position, with our data showing considerable correspondence to post-mortem myeloarchitectonic studies, both in cross-participant averages and in individuals. The core region contains two ‘mirror-image‘ tonotopic maps oriented along the same axis as observed in macaque and owl monkey. We suggest that thee two maps within the core are the human analogues of primate auditory areas A1 and R. The core occupies a much smaller portion of tonotopically organized cortex on the superior temporal plane and gyrus than is generally supposed. The multi-modal approach to defining the auditory core will facilitate investigations of structure-function relationships, comparative neuroanatomical studies, and promises new biomarkers for diagnosis and clinical studies.

Dick, Frederic; Tierney, Adam Taylor; Lutti, Antoine; Josephs, Oliver; Sereno, Martin I.; Weiskopf, Nikolaus

2012-01-01

265

[Identification technique for in vivo ingredients of traditional Chinese medicines based on LC-MS analysis].  

PubMed

Serum pharmacochemistry of traditional Chinese medicine (TCM) is a direct and effective method for determining efficacious substance foundation of TCM. However, the complexity of chemical constituents of TCM and the interaction among ingredients in the in vitro process make the analysis on in vitro ingredients of TCM arduous. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become the cornerstone in detection and characterization of in vivo ingredients of TCM because of its sensitivity and ability to analyze complex mixtures. However, due to significant interference from endogenous species, detection and identification of the constituents of TCM in the biological matrices are often difficult. There is a crying need for introducing specialized ingredient identification techniques to avoid artificial omission of in vivo ingredients of TCM. On the basis of the analysis on transitional ingredients in rat blood, this essay introduces the application of such pattern recognition methods as mass defect filter, Metabolynx software and principal component analysis, partial least squared discriminant analysis and orthogonal partial least squared discriminant analysis in identifying in vivo ingredients of TCM. PMID:22997821

Yan, Guangli; Hang, Ying; Wang, Xijun

2012-06-01

266

In vitro gene regulatory networks predict in vivo function of liver  

Microsoft Academic Search

BACKGROUND: Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed. RESULTS: Here we use the

Youping Deng; David R Johnson; Xin Guan; Choo Y Ang; Junmei Ai; Edward J Perkins

2010-01-01

267

Exposure to low mercury concentration in vivo impairs myocardial contractile function  

SciTech Connect

Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl{sub 2} (1st dose 4.6 {mu}g/kg, subsequent dose 0.07 {mu}g/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and {beta}-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na{sup +}-K{sup +} ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and {alpha}-1 isoform of NKA, whereas {alpha}-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: > Unchanges blood pressure, heart rate, systolic pressure. > Increases end diastolic pressure. > Promotes cardiac contractility dysfunction. > Decreases NKA activity, NCX and SERCA, increases PLB protein expression. > Small concentrations constitutes environmental cardiovascular risk factor.

Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Bartolome, Maria Visitacion [Department of Physiology, Universidad Complutense de Madrid (Spain); Fernandes, Aurelia Araujo [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Cachofeiro, Victoria; Lahera, Vicente [Department of Physiology, Universidad Complutense de Madrid (Spain); Salaices, Mercedes [Department of Pharmacology, Universidad Autonoma de Madrid (Spain); Stefanon, Ivanita [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Vassallo, Dalton Valentim, E-mail: daltonv2@terra.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Health Science Center of Vitoria-EMESCAM, Vitoria, ES (Brazil)

2011-09-01

268

Nobiletin, a citrus polymethoxyflavonoid, suppresses multiple angiogenesis-related endothelial cell functions and angiogenesis in vivo.  

PubMed

Nobiletin is a citrus polymethoxyflavonoid that suppresses tumor growth and metastasis, both of which depend on angiogenesis. We recently identified nobiletin as a cell differentiation modulator. Because cell differentiation is a critical event in angiogenesis, it might be possible that nobiletin could exhibit antiangiogenic activity, resulting in suppression of these tumor malignant properties. To verify this possibility, we examined the antiangiogenic effects of nobiletin in vitro and in vivo. Nobiletin had concentration-dependent inhibitory effects on multiple functions of angiogenesis-related endothelial cells (EC); it suppressed the proliferation, migration and tube formation on matrigel of human umbilical vein EC (HUVEC) stimulated with endothelial cell growth supplement (ECGS), a mixture of acidic and basic fibroblast growth factors (FGFs). Gelatin zymography and northern blotting revealed that nobiletin suppressed pro-matrix metalloproteinase-2 (proMMP-2) production and MMP-2 mRNA expression in ECGS-stimulated HUVEC. Nobiletin also downregulated cell-associated plasminogen activator (PA) activity and urokinase-type PA mRNA expression. Furthermore, nobiletin inhibited angiogenic differentiation induced by vascular endothelial growth factor and FGF, an in vitro angiogenesis model. This inhibition was accompanied by downregulation of angiogenesis-related signaling molecules, such as extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase, and transcriptional factors (c-Jun and signal transducer and activator of transcription 3), and activation of the caspase pathway. In a chick embryo chorioallantoic membrane assay, nobiletin showed an antiangiogenic activity, the ID(50) value being 10?g (24.9nmol) per egg. These results indicate that nobiletin is a novel antiangiogenic compound that exhibits its activity through combined inhibition of multiple angiogenic EC functions. PMID:20670297

Kunimasa, Kazuhiro; Ikekita, Masahiko; Sato, Mayumi; Ohta, Toshiro; Yamori, Yukio; Ikeda, Megumi; Kuranuki, Sachi; Oikawa, Tsutomu

2010-11-01

269

Clinical Application of Functional Analysis Methodology  

PubMed Central

Functional analysis (FA) methodology is a well-established standard for assessment in applied behavior analysis research. Although used less commonly in clinical (nonresearch) application, the basic components of an FA can be adapted easily in many situations to facilitate the treatment of problem behavior. This article describes practical aspects of FA methodology and suggests ways that it can be incorporated into routine clinical work.

Iwata, Brian A; Dozier, Claudia L

2008-01-01

270

A factor analysis model for functional genomics  

PubMed Central

Background Expression array data are used to predict biological functions of uncharacterized genes by comparing their expression profiles to those of characterized genes. While biologically plausible, this is both statistically and computationally challenging. Typical approaches are computationally expensive and ignore correlations among expression profiles and functional categories. Results We propose a factor analysis model (FAM) for functional genomics and give a two-step algorithm, using genome-wide expression data for yeast and a subset of Gene-Ontology Biological Process functional annotations. We show that the predictive performance of our method is comparable to the current best approach while our total computation time was faster by a factor of 4000. We discuss the unique challenges in performance evaluation of algorithms used for genome-wide functions genomics. Finally, we discuss extensions to our method that can incorporate the inherent correlation structure of the functional categories to further improve predictive performance. Conclusion Our factor analysis model is a computationally efficient technique for functional genomics and provides a clear and unified statistical framework with potential for incorporating important gene ontology information to improve predictions.

Kustra, Rafal; Shioda, Romy; Zhu, Mu

2006-01-01

271

Efficient inhibition of miR-155 function in vivo by peptide nucleic acids  

PubMed Central

MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.

Fabani, Martin M.; Abreu-Goodger, Cei; Williams, Donna; Lyons, Paul A.; Torres, Adrian G.; Smith, Kenneth G. C.; Enright, Anton J.; Gait, Michael J.; Vigorito, Elena

2010-01-01

272

Transcriptome analysis of Traf6 function in the innate immune response of zebrafish embryos  

Microsoft Academic Search

TRAF6 is a key player at the cross-roads of development and immunity. The analysis of its in vivo molecular function is a great challenge since severe developmental defects and early lethality caused by Traf6 deficiency in knock-out mice interfere with analyses of the immune response. In this study we have used a new strategy to analyze the function of Traf6

Oliver W. Stockhammer; Han Rauwerda; Floyd R. Wittink; Timo M. Breit; Annemarie H. Meijer; Herman P. Spaink

2010-01-01

273

In vivo Analysis of Tissue Response to Plasma-Treated Collagen-I-Coated Titanium Alloys  

Microsoft Academic Search

Purpose: The aim of this study was to evaluate the in vivo tissue response to low-pressure plasma-pretreated collagen-I-coated titanium implant in a middle-term mouse model. Methods: Plasma-treated collagen-coated titanium implants were transplanted into the dorsal skinfold chambers of BALB\\/c mice. Untreated, regular titanium implant material served as control. The neovascularization (functional vessel density) of the implant border zone and of

J. Hauser; A. Ring; A. Schaffran; L. Henrich; S. A. Esenwein; H. U. Steinau; I. Stricker; S. Langer

2009-01-01

274

In vivo coassembly of a divergent beta-tubulin subunit (c beta 6) into microtubules of different function  

PubMed Central

alpha- and beta-Tubulin are encoded in vertebrate genomes by a family of approximately 6-7 functional genes whose polypeptide products differ in amino acid sequence. In the chicken, one beta-tubulin isotype (c beta 6) has previously been found to be expressed only in thrombocytes and erythroid cells, where it is assembled into a circumferential ring of marginal band microtubules. In light of its unique in vivo utilization and its divergent assembly properties in vitro, we used DNA transfection to test whether this isotype could be assembled in vivo into microtubules of divergent functions. Using an antibody specific to c beta 6, we have found that upon transfection this polypeptide is freely coassembled into an extensive array of interphase cytoplasmic microtubules and into astral and pole-to-chromosome or pole-to-pole microtubules during mitosis. Further, examination of developing chicken erythrocytes reveals that both beta-tubulins that are expressed in these cells (c beta 6 and c beta 3) are found as co-polymers of the two isoforms. These results, in conjunction with efforts that have localized various other beta-tubulin isotypes, demonstrate that to the resolution limit afforded by light microscopy in vivo microtubules in vertebrates are random copolymers of available isotypes. Although these findings are consistent with functional interchangeability of beta- tubulin isotypes, we have also found that in vivo microtubules enriched in c beta 3 polypeptides are more sensitive to cold depolymerization than those enriched in c beta 6. This differential quantitative utilization of the two endogenous isotypes documents that some in vivo functional differences between isotypes do exist.

1987-01-01

275

Isolation and functional characterization of human erythroblasts at distinct stages: implications for understanding of normal and disordered erythropoiesis in vivo.  

PubMed

Terminal erythroid differentiation starts from morphologically recognizable proerythroblasts that proliferate and differentiate to generate red cells. Although this process has been extensively studied in mice, its characterization in humans is limited. By examining the dynamic changes of expression of membrane proteins during in vitro human terminal erythroid differentiation, we identified band 3 and ?4 integrin as optimal surface markers for isolating 5 morphologically distinct populations at successive developmental stages. Functional analysis revealed that these purified cell populations have distinct mitotic capacity. Use of band 3 and ?4 integrin enabled us to isolate erythroblasts at specific developmental stages from primary human bone marrow. The ratio of erythroblasts at successive stages followed the predicted 1:2:4:8:16 pattern. In contrast, bone marrows from myelodysplastic syndrome patients exhibited altered terminal erythroid differentiation profiles. Thus, our findings not only provide new insights into the genesis of the red cell membrane during human terminal erythroid differentiation but also offer a means of isolating and quantifying each developmental stage during terminal erythropoiesis in vivo. Our findings should facilitate a comprehensive cellular and molecular characterization of each specific developmental stage of human erythroblasts and should provide a powerful means of identifying stage-specific defects in diseases associated with pathological erythropoiesis. PMID:23422750

Hu, Jingping; Liu, Jing; Xue, Fumin; Halverson, Gregory; Reid, Marion; Guo, Anqi; Chen, Lixiang; Raza, Azra; Galili, Naomi; Jaffray, Julie; Lane, Joseph; Chasis, Joel Anne; Taylor, Naomi; Mohandas, Narla; An, Xiuli

2013-02-19

276

Functional graphene oxide as a plasmid-based Stat3 siRNA carrier inhibits mouse malignant melanoma growth in vivo  

NASA Astrophysics Data System (ADS)

Graphene oxide (GO) has attracted intensive interest in the biomedical field in recent years. We investigate whether the use of functional graphene oxide as an efficient delivery system for delivering specific molecular antitumor therapeutics in vivo could achieve a more excellent antitumor effect. Constitutive activation of signal transducer and activator of transcription 3 (Stat3) promotes survival in a wide spectrum of human cancers. In this paper, we study the in vivo behavior of graphene oxide chemically functionalized with polyethylenimine and polyethylene glycol (GO-PEI-PEG) as a plasmid-based Stat3-specific small interfering RNA (siRNA) carrier in mouse malignant melanoma. The in vivo results indicate significant regression in tumor growth and tumor weight after plasmid-based Stat3 siRNA delivered by GO-PEI-PEG treatment. Moreover, there was no significant side effect from GO-PEI-PEG treatment according to histological examination and blood chemistry analysis in mice. Thus, our work is the first success of using GO-PEI-PEG as a promising carrier for plasmid Stat3 siRNA delivery and down-regulation of Stat3 by a polymer-mediated vehicle and suggests the great promise of graphene in biomedical applications such as cancer treatment.

Yin, Di; Li, Yang; Lin, Hang; Guo, Baofeng; Du, Yanwei; Li, Xin; Jia, Huijie; Zhao, Xuejian; Tang, Jun; Zhang, Ling

2013-03-01

277

In vivo mutation analysis using the ?X174 transgenic mouse and comparisons with other transgenes and endogenous genes  

Microsoft Academic Search

The ?X174 transgenic mouse was first developed as an in vivo Ames test, detecting base pair substitution (bps) at a single bp in a reversion assay. A forward mutational assay was also developed, which is a gain of function assay that also detects bps exclusively. Later work with both assays focused on establishing that a mutation was fixed in vivo

Carrie R. Valentine; Robert R. Delongchamp; Mason G. Pearce; Heather F. Rainey; Vasily N. Dobrovolsky; Heinrich V. Malling; Robert H. Heflich

2010-01-01

278

The functional analysis of problematic verbal behavior  

PubMed Central

This study describes procedures and outcomes in a functional analysis of problem behavior of 2 public school students. For a 13-year-old honors student, bizarre tacts (labeled as psychotic speech by school staff) were maintained by attention. For a 15-year-old with autism, the functional analysis revealed that perseverative mands for toileting were controlled by attention; mands for edible items were controlled by access to any food item; and mands for nonedible items were maintained by access to the specific item manded. The “problematic” aspects of the verbal behavior differed—the bizarre speech was problematic based on its content, but the perseverative verbalizations resulted in high response cost for classroom staff. Research in the area of problematic verbal behavior is sparse and warrants further attention from behavior analysts who work in public school settings. This research demonstrates the applicability and relevance of functionally analyzing problematic verbal behavior in public school settings.

Ewing, Christopher B.; Magee, Sandy K.; Ellis, Janet

2002-01-01

279

Quantitative analysis of gene function in the Drosophila embryo.  

PubMed Central

The specific functions of gene products frequently depend on the developmental context in which they are expressed. Thus, studies on gene function will benefit from systems that allow for manipulation of gene expression within model systems where the developmental context is well defined. Here we describe a system that allows for genetically controlled overexpression of any gene of interest under normal physiological conditions in the early Drosophila embryo. This regulated expression is achieved through the use of Drosophila lines that express a maternal mRNA for the yeast transcription factor GAL4. Embryos derived from females that express GAL4 maternally activate GAL4-dependent UAS transgenes at uniform levels throughout the embryo during the blastoderm stage of embryogenesis. The expression levels can be quantitatively manipulated through the use of lines that have different levels of maternal GAL4 activity. Specific phenotypes are produced by expression of a number of different developmental regulators with this system, including genes that normally do not function during Drosophila embryogenesis. Analysis of the response to overexpression of runt provides evidence that this pair-rule segmentation gene has a direct role in repressing transcription of the segment-polarity gene engrailed. The maternal GAL4 system will have applications both for the measurement of gene activity in reverse genetic experiments as well as for the identification of genetic factors that have quantitative effects on gene function in vivo.

Tracey, W D; Ning, X; Klingler, M; Kramer, S G; Gergen, J P

2000-01-01

280

Fluorescence spectroscopic analysis (FSA) detects quantitative changes in atherosclerotic plaque collagen and elastin content In Vivo  

NASA Astrophysics Data System (ADS)

In order to assess the capacity for in vivo fluorescence spectroscopi8c analysis of arterial collagen and elastin, fluorescence emission intensity was recorded form rabbit aorta after angioplasty and stent implant, and correlated with extracted elastin and collagen content. FEI from saline treated rabbits after stent implant was higher between 485 and 500 nm than after anti-inflammatory treatment. FEI was significantly decreased after implantation of shorter stents at 476-500 nm. Multiple regression analysis demonstrated an excellent correlation between FEI and elastin and HPLC- measured collagen content at 486-500 nm and 476-480 nm respectively. Conclusions: FEI recorded in vivo form arterial intimal surface, can be successfully used for quantitative assessment of compositional changes in connective tissue. Stent implant can induce changes in intimal arterial structure at discrete sites distant from the stent implant site.

Christov, Alexander M.; Dai, Erbin; Liu, Liying; Guan, Haiyan; Bernards, Mark A.; Cavers, Paul B.; Susko, David; Lucas, Alexandra

2002-05-01

281

Prompt-gamma neutron activation analysis facility for in vivo body composition studies in small animals.  

PubMed

The design, calibration, dosimetry and performance evaluation of a prompt-gamma neutron activation analysis facility for in vivo body composition studies in small animals (i.e. rats or rabbits) is discussed. The system design was guided by Monte Carlo transport calculations using MCNP-4C code. A system was built and performance evaluation was made using a 185-GBq Pu-Be neutron source. Prompt-gamma rays produced by neutron capture reactions were detected by a combination of a NaI(Tl) scintillation and a HPGe semiconductor detectors. Nitrogen and chlorine were quantified by analysis of the 10.83-MeV and 6.11-MeV peaks, respectively. Appropriate corrections for the animal body size were determined. The facility described allows the in vivo determination of protein and extracellular space in sets of experimental animals. PMID:14762643

Stamatelatos, I E; Kasviki, K; Green, S; Gainey, M; Kalef-Ezra, J; Beddoe, A

2004-02-05

282

The feasibility of accelerator-based in vivo neutron activation analysis of nitrogen  

Microsoft Academic Search

The feasibility of accelerator-based in vivo neutron activation analysis of nitrogen has been investigated. It was found that a moderated neutron flux from ?10?A of 2.5MeV protons on a 9Be target performed as well as, and possibly slightly better than the existing isotope-based approach in terms of net counts per unit subject dose. Such a system may be an attractive

J. M. O’Meara; B. W. Blackburn; D. L. Chichester; D. P. Gierga; J. C. Yanch

2001-01-01

283

Application of Electrical Stimulation for Functional Tissue Engineering In Vitro and In Vivo.  

National Technical Information Service (NTIS)

The present invention provides new methods for the in vitro preparation of bioartificial tissue equivalents and their enhanced integration after implantation in vivo. These methods include submitting a tissue construct to a biomimetic electrical stimulati...

G. V. Novakovic H. Park L. Freed M. Radisic R. Langer

2004-01-01

284

In vivo function of airway epithelial TLR2 in host defense against bacterial infection  

PubMed Central

Decreased Toll-like receptor 2 (TLR2) expression has been reported in patients with chronic obstructive pulmonary disease and in a murine asthma model, which may predispose the hosts to bacterial infections, leading to disease exacerbations. Since airway epithelial cells serve as the first line of respiratory mucosal defense, the present study aimed to reveal the role of airway epithelial TLR2 signaling to lung bacterial [i.e., Mycoplasma pneumoniae (Mp)] clearance. In vivo TLR2 gene transfer via intranasal inoculation of adenoviral vector was performed to reconstitute TLR2 expression in airway epithelium of TLR2?/? BALB/c mice, with or without ensuing Mp infection. TLR2 and lactotransferrin (LTF) expression in airway epithelial cells and lung Mp load were assessed. Adenovirus-mediated TLR2 gene transfer to airway epithelial cells of TLR2?/? mice reconstituted 30–40% TLR2 expression compared with TLR2+/+ cells. Such airway epithelial TLR2 reconstitution in TLR2?/? mice significantly reduced lung Mp load (an appropriate 45% reduction), coupled with elevated LTF expression. LTF expression in mice was shown to be mainly dependent on TLR2 signaling in response to Mp infection. Exogenous human LTF protein dose-dependently decreased lung bacterial load in Mp-infected TLR2?/? mice. In addition, human LTF protein directly dose-dependently decreased Mp levels in vitro. These data indicate that reconstitution of airway epithelial TLR2 signaling in TLR2?/? mice significantly restores lung defense against bacteria (e.g., Mp) via increased lung antimicrobial protein LTF production. Our findings may offer a deliverable approach to attenuate bacterial infections in airways of asthma or chronic obstructive pulmonary disease patients with impaired TLR2 function.

Wu, Qun; Jiang, Di; Minor, Maisha N.; Martin, Richard J.

2011-01-01

285

A Surface Groove Essential for Viral Bcl-2 Function During Chronic Infection In Vivo  

PubMed Central

Antiapoptotic Bcl-2 family proteins inhibit apoptosis in cultured cells by binding BH3 domains of proapoptotic Bcl-2 family members via a hydrophobic BH3 binding groove on the protein surface. We investigated the physiological importance of the BH3 binding groove of an antiapoptotic Bcl-2 protein in mammals in vivo by analyzing a viral Bcl-2 family protein. We show that the ?-herpesvirus 68 (?HV68) Bcl-2 family protein (?HV68 v-Bcl-2), which is known to inhibit apoptosis in cultured cells, inhibits both apoptosis in primary lymphocytes and Bax toxicity in yeast. Nuclear magnetic resonance determination of the ?HV68 v-Bcl-2 structure revealed a BH3 binding groove that binds BH3 domain peptides from proapoptotic Bcl-2 family members Bax and Bak via a molecular mechanism shared with host Bcl-2 family proteins, involving a conserved arginine in the BH3 peptide binding groove. Mutations of this conserved arginine and two adjacent amino acids to alanine (SGR to AAA) within the BH3 binding groove resulted in a properly folded protein that lacked the capacity of the wild-type ?HV68 v-Bcl-2 to bind Bax BH3 peptide and to block Bax toxicity in yeast. We tested the physiological importance of this v-Bcl-2 domain during viral infection by engineering viral mutants encoding a v-Bcl-2 containing the SGR to AAA mutation. This mutation resulted in a virus defective for both efficient reactivation of ?HV68 from latency and efficient persistent ?HV68 replication. These studies demonstrate an essential functional role for amino acids in the BH3 peptide binding groove of a viral Bcl-2 family member during chronic infection.

Petros, Andrew M; Nettesheim, David; van Dyk, Linda F.; Labrada, Lucia; Speck, Samuel H; Levine, Beth

2005-01-01

286

In vivo functional assay of a recombinant aquaporin in Pichia pastoris.  

PubMed

The water channel protein PvTIP3;1 (alpha-TIP) is a member of the major intrinsic protein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeast Pichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-type P. pastoris displayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts of P. pastoris expressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of the Escherichia coli glycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity. PMID:16461705

Daniels, Mark J; Wood, Malcolm R; Yeager, Mark

2006-02-01

287

The NFIII/OCT-1 binding site stimulates adenovirus DNA replication in vivo and is functionally redundant with adjacent sequences.  

PubMed Central

The inverted terminal repeat (ITR) of adenovirus type 5 (Ad5) is 103 bp in length and contains the origin of DNA replication. Cellular transcription factors NFI/CTF and NFIII/OCT-1 bind to sites within the ITR and participate in the initiation of viral DNA replication in vitro. The ITR also contains multiple copies of two conserved sequence motifs that bind the cellular transcription factors SP1 and ATF. We have analyzed a series of viruses that carry deletions at the left terminus of Ad5. A virus carrying a deletion of the NFIII/OCT-1, SP1, and ATF sites within the ITR (mutant dl309-44/107) was wild type for virus growth. However, the deletion of these elements in addition to sequences immediately flanking the ITR (mutant dl309-44/195) resulted in a virus that grew poorly. The analysis of growth parameters of these and other mutants demonstrate that the NFIII/OCT-1 and adjacent SP1 sites augment the accumulation of viral DNA following infection. The function of these elements was most evident in coinfections with a wild-type virus, suggesting that these sites enhance the ability of a limiting trans-acting factor(s), that stimulates viral DNA replication, to interact with the ITR. The results of these analyses indicate functional redundancy between different transcription elements at the left terminus of the Ad5 genome and demonstrate that the NFIII/OCT-1 site and adjacent SP1 site, previously thought to be nonessential for adenovirus growth, play a role in viral DNA replication in vivo. Images

Hatfield, L; Hearing, P

1993-01-01

288

Genetic analysis of glutamatergic function in Drosophila  

SciTech Connect

Neurotransmitters are essential for communication between neurons and hence are vital in the overall integrative functioning of the nervous system. Previous work on acetylcholine metabolism in the fruit fly, Drosophila melanogaster, has also raised the possibility that transmitter metabolism may play a prominent role in either the achievement or maintenance of the normal structure of the central nervous system in this species. Unfortunately, acetylcholine is rather poorly characterized as a neurotransmitter in Drosophila; consequently, we have begun an analysis of the role of glutamate (probably the best characterized transmitter in this organism) in the formation and/or maintenance of nervous system structure. We present here the results of a series of preliminary analyses. To suggest where glutamatergic function may be localized, an examination of the spatial distribution of high affinity (/sup 3/H)-glutamate binding sites are presented. We present the results of an analysis of the spatial and temporal distribution of enzymatic activities thought to be important in the regulation of transmitter-glutamate pools (i.e., glutamate oxaloacetic transaminase, glutaminase, and glutamate dehydrogenase). To begin to examine whether mutations in any of these functions are capable of affecting glutamatergic activity, we present the results of an initial genetic analysis of one enzymatic function, glutamate oxaloacetic transaminase (GOT), chosen because of its differential distribution within the adult central nervous system and musculature.

Chase, B.A.; Kankel, D.R.

1987-01-01

289

First in vivo evidence for a functional interaction between chemokine and cannabinoid systems in the brain.  

PubMed

Growing evidence supports the idea that in addition to their well established role in the immune system, chemokines might play a role in both normal and pathological brain function, and the chemokine network could interact with other neuromodulators. The chemokine stromal cell-derived growth factor (SDF)-1alpha/CXCL12, a member of the CXC chemokine family, was tested for its possible effect on the analgesic responses of the cannabinoid receptor agonist aminoalkylindole 4,5-dihydro-2-methyl-4-(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo-[3,2,1ij]quinolin-6-one [(+)-WIN 55,212-2, hereafter WIN 55,212-2] at the level of the periaqueductal gray (PAG), a brain region critical to the processing of pain signals, and a primary site of action of many analgesic compounds. The administration of WIN 55,212-2 (0.1-0.4 microg/microl) into the PAG resulted in antinociception in a dose-dependent manner. The selective cannabinoid (CB)1 antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A; 1-10 microg) given into the PAG blocked the WIN 55,212-2-induced antinociception. In contrast, the selective CB2 antagonist N-[(1S)-endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 10 microg) did not alter the WIN 55,212-2-induced antinociception. Pretreatment with SDF-1alpha/CXCL12 (100 ng) caused a reduction in antinociceptive responses of WIN 55,212-2. The inhibitory effect of SDF-1alpha/CXCL12 on WIN 55,212-2-induced antinociception was reversed by octahydrochloride [corrected] hydrate (AMD 3100) (10-50 ng), an antagonist of the SDF-1alpha/CXCL12, acting at its receptor, CXCR4. This study reports the first in vivo evidence of a functional interaction between chemokine and cannabinoid systems in the brain, showing that the activation of SDF-1alpha/CXCL12 receptors (CXCR4) in the PAG interferes with the analgesic effects of WIN 55212-2. PMID:18281594

Benamar, Khalid; Geller, Ellen B; Adler, Martin W

2008-02-15

290

In vivo stem cell function of interleukin-3-induced blast cells  

SciTech Connect

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, the authors obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. They examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 {times} 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 {times} 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.

Tsunoda, J.; Okada, S.; Suda, J.; Nagayoshi, K.; Nakauchi, H.; Hatake, K.; Miura, Y.; Suda, T. (Department of Medicine, Jichi Medical School, Tochigi-ken (Japan))

1991-07-15

291

Two Classes of Boolean Functions for Dependency Analysis  

Microsoft Academic Search

Many static analyses for declarative programming\\/database languages use Boolean functions to express dependencies among variables or argument positions. Examples include groundness analysis, arguably the most important analysis for logic programs, finiteness analysis and functional dependency analysis for databases. We identify two classes of Boolean functions that have been used: positive and definite functions, and we systematically investigate these classes and

Tania Armstrong; Kim Marriott; Peter Schachte; Harald Sřndergaard

1998-01-01

292

Design and analysis of a novel mechanical loading machine for dynamic in vivo axial loading  

PubMed Central

This paper describes the construction of a loading machine for performing in vivo, dynamic mechanical loading of the rodent forearm. The loading machine utilizes a unique type of electromagnetic actuator with no mechanically resistive components (servotube), allowing highly accurate loads to be created. A regression analysis of the force created by the actuator with respect to the input voltage demonstrates high linear correlation (R2 = 1). When the linear correlation is used to create dynamic loading waveforms in the frequency (0.5–10 Hz) and load (1–50 N) range used for in vivo loading, less than 1% normalized root mean square error (NRMSE) is computed. Larger NRMSE is found at increased frequencies, with 5%–8% occurring at 40 Hz, and reasons are discussed. Amplifiers (strain gauge, linear voltage displacement transducer (LVDT), and load cell) are constructed, calibrated, and integrated, to allow well-resolved dynamic measurements to be recorded at each program cycle. Each of the amplifiers uses an active filter with cutoff frequency at the maximum in vivo loading frequencies (50 Hz) so that electronic noise generated by the servo drive and actuator are reduced. The LVDT and load cell amplifiers allow evaluation of stress-strain relationships to determine if in vivo bone damage is occurring. The strain gauge amplifier allows dynamic force to strain calibrations to occur for animals of different sex, age, and strain. Unique features are integrated into the loading system, including a weightless mode, which allows the limbs of anesthetized animals to be quickly positioned and removed. Although the device is constructed for in vivo axial bone loading, it can be used within constraints, as a general measurement instrument in a laboratory setting.

Macione, James; Nesbitt, Sterling; Pandit, Vaibhav; Kotha, Shiva

2012-01-01

293

Protective effect of trimetazidine on myocardial mitochondrial function in an ex-vivo model of global myocardial ischemia  

Microsoft Academic Search

Trimetazidine is an anti-ischemic drug whose cytoprotective mechanisms are not yet fully understood (but until now mainly related to the trimetazidine-induced “metabolic shift” from lipid ?-oxidation to glucose aerobic oxidation). We studied the effect of trimetazidine on the mitochondrial function of ischemic Wistar rat hearts perfused with glucose, using a model of ex-vivo perfusion (Langendorff system). We measured the electrical

Pedro Monteiro; Ana I. Duarte; Lino M. Gonçalves; António Moreno; Luís A. Providęncia

2004-01-01

294

In vivo assembly of functional U7 snRNP requires RNA backbone flexibility within the Sm-binding site  

Microsoft Academic Search

Most histone precursor mRNAs (pre-mRNAs) in metazoans are matured by 3?-end cleavage directed by the U7 small nuclear ribonucleoprotein (snRNP). RNA functional groups necessary for in vivo assembly and activity of the U7 snRNP were examined by nucleotide-analog interference mapping and mutagenesis using a chimeric mouse histone H4 pre-mRNA–U7 snRNA construct that is cleaved in cis in Xenopus laevis oocytes.

Nikolay G Kolev; Joan A Steitz

2006-01-01

295

Fracture Analysis of Functionally Graded Materials  

NASA Astrophysics Data System (ADS)

This paper reports our recent research works on crack analysis in continuously non-homogeneous and linear elastic functionally graded materials. A meshless boundary element method is developed for this purpose. Numerical examples are presented and discussed to demonstrate the efficiency and the accuracy of the present numerical method, and to show the effects of the material gradation on the crack-opening-displacements and the stress intensity factors.

Zhang, Ch.; Gao, X. W.; Sladek, J.; Sladek, V.

2010-05-01

296

In vivo neutron activation analysis: body composition studies in health and disease  

SciTech Connect

In vivo analysis of body elements by neutron activation is an important tool in medical research. It has provided a direct quantitative measure of body composition of human beings in vivo. Basic physiological differences related to age, sex, race, and body size have been assessed by this noninvasive technique. The diagnosis and management of patients with various metabolic disorders and diseases has also been demonstrated. Two major facilities at Brookhaven are being utilized exclusively for in vivo neutron activation analysis (IVNAA) of calcium, phosphorus, sodium, chlorine, nitrogen, hydrogen, and potassium. These elements serve as the basis for a four compartment model of body composition: protein, water, mineral ash, and fat. Variations in these compartments are demonstrated in clinical research programs investigating obesity, anorexia, cancer, renal failure, osteoporosis, and normal aging. IVNAA continues to provide a unique approach to the evaluation of clinical diagnosis, efficacy of therapeutic regimens, and monitoring of the aging process. Classical balance studies usually require the patient to be admitted to a hospital for extended periods of confinement. IVNAA, however, allows for clinical management of the patient on an out-patient basis, an important aspect for treatment of chronic diseases. 25 references, 3 figures, 5 tables.

Ellis, K.J.; Cohn, S.H.

1984-01-01

297

Compound ex vivo and in silico method for hemodynamic analysis of stented arteries.  

PubMed

Hemodynamic factors such as low wall shear stress have been shown to influence endothelial healing and atherogenesis in stent-free vessels. However, in stented vessels, a reliable quantitative analysis of such relations has not been possible due to the lack of a suitable method for the accurate acquisition of blood flow. The objective of this work was to develop a method for the precise reconstruction of hemodynamics and quantification of wall shear stress in stented vessels. We have developed such a method that can be applied to vessels stented in or ex vivo and processed ex vivo. Here we stented the coronary arteries of ex vivo porcine hearts, performed vascular corrosion casting, acquired the vessel geometry using micro-computed tomography and reconstructed blood flow and shear stress using computational fluid dynamics. The method yields accurate local flow information through anatomic fidelity, capturing in detail the stent geometry, arterial tissue prolapse, radial and axial arterial deformation as well as strut malapposition. This novel compound method may serve as a unique tool for spatially resolved analysis of the relationship between hemodynamic factors and vascular biology. It can further be employed to optimize stent design and stenting strategies. PMID:23516442

Rikhtegar, Farhad; Pacheco, Fernando; Wyss, Christophe; Stok, Kathryn S; Ge, Heng; Choo, Ryan J; Ferrari, Aldo; Poulikakos, Dimos; Müller, Ralph; Kurtcuoglu, Vartan

2013-03-13

298

Structural and Functional Analysis of Saccharomyces Cerevisiae Mob1  

SciTech Connect

The Mob proteins function as activator subunits for the Dbf2/Dbf20 family of protein kinases. Human and Xenopus Mob1 protein structures corresponding to the most conserved C-terminal core, but lacking the variable N-terminal region, have been reported and provide a framework for understanding the mechanism of Dbf2/Dbf20 regulation. Here, we report the 2.0 {angstrom} X-ray crystal structure of Saccharomyces cerevisiae Mob1 containing both the conserved C-terminal core and the variable N-terminal region. Within the N-terminal region, three novel structural elements are observed; namely, an {alpha}-helix denoted H0, a strand-like element denoted S0 and a short {beta} strand denoted S-1. Helix H0 associates in an intermolecular manner with a second Mob1 molecule to form a Mob1 homodimer. Strand S0 binds to the core domain in an intramolecular manner across a putative Dbf2 binding site mapped by Mob1 temperature-sensitive alleles and NMR binding experiments. In vivo functional analysis demonstrates that Mob1 mutants that target helix H0 or its reciprocal binding site are biologically compromised. The N-terminal region of Mob1 thus contains structural elements that are functionally important.

Mrkobrada,S.; Boucher, L.; Tyers, D.; Sicheri, F.

2006-01-01

299

Structure-function analysis of the trypanosomatid spliced leader RNA.  

PubMed Central

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.

Goncharov, I; Xu, Y X; Zimmer, Y; Sherman, K; Michaeli, S

1998-01-01

300

Ex vivo characterization of human CD8+ T subsets with distinct replicative history and partial effector functions.  

PubMed

After antigenic challenge, naive T lymphocytes enter a program of proliferation and differentiation during the course of which they acquire effector functions and may ultimately become memory cells. In humans, the pathways of effector and memory T-cell differentiation remain poorly defined. Here we describe the properties of 2 CD8+ T-lymphocyte subsets, RA+CCR7-27+28+ and RA+CCR7-27+28-, in human peripheral blood. These cells display phenotypic and functional features that are intermediate between naive and effector T cells. Like naive T lymphocytes, both subsets show relatively long telomeres. However, unlike the naive population, these T cells exhibit reduced levels of T-cell receptor excision circles (TRECs), indicating they have undergone additional rounds of in vivo cell division. Furthermore, we show that they also share effector-type properties. At equivalent in vivo replicative history, the 2 subsets express high levels of Fas/CD95 and CD11a, as well as increasing levels of effector mediators such as granzyme B, perforin, interferon gamma, and tumor necrosis factor alpha. Both display partial ex vivo cytolytic activity and can be found among cytomegalovirus-specific cytolytic T cells. Taken together, our data point to the presence of T cells with intermediate effector-like functions and suggest that these subsets consist of T lymphocytes that are evolving toward a more differentiated effector or effector-memory stage. PMID:12750165

Rufer, Nathalie; Zippelius, Alfred; Batard, Pascal; Pittet, Mikael J; Kurth, Isabel; Corthesy, Patricia; Cerottini, Jean-Charles; Leyvraz, Serge; Roosnek, Eddy; Nabholz, Markus; Romero, Pedro

2003-05-15

301

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo  

PubMed Central

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer.

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; H?yer-Hansen, Gunilla

2012-01-01

302

Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex  

PubMed Central

The Drosophila Glued gene product shares sequence homology with the p150 component of vertebrate dynactin. Dynactin is a multiprotein complex that stimulates cytoplasmic dynein-mediated vesicle motility in vitro. In this report, we present biochemical, cytological, and genetic evidence that demonstrates a functional similarity between the Drosophila Glued complex and vertebrate dynactin. We show that, similar to the vertebrate homologues in dynactin, the Glued polypeptides are components of a 20S complex. Our biochemical studies further reveal differential expression of the Glued polypeptides, all of which copurify as microtubule-associated proteins. In our analysis of the Glued polypeptides encoded by the dominant mutation, Glued, we identify a truncated polypeptide that fails to assemble into the wild-type 20S complex, but retains the ability to copurify with microtubules. The spatial and temporal distribution of the Glued complex during oogenesis is shown by immunocytochemistry methods to be identical to the pattern previously described for cytoplasmic dynein. Significantly, the pattern of Glued distribution in oogenesis is dependent on dynein function, as well as several other gene products known to be required for proper dynein localization. In genetic complementation studies, we find that certain mutations in the cytoplasmic dynein heavy chain gene Dhc64C act as dominant suppressors or enhancers of the rough eye phenotype of the dominant Glued mutation. Furthermore, we show that a mutation that was previously isolated as a suppressor of the Glued mutation is an allele of Dhc64C. Together with the observed dependency of Glued localization on dynein function, these genetic interactions demonstrate a functional association between the Drosophila dynein motor and Glued complexes.

1995-01-01

303

Geometric modeling, functional parameter calculation, and visualization of the in-vivo distended rectal wall  

NASA Astrophysics Data System (ADS)

The rectum can distend to accommodate stool, and contracts in response to distention during defecation. Rectal motor dysfunctions are implicated in the pathophysiology of functional defecation disorders and fecal incontinence. These rectal motor functions can be studied by intra-luminal measurements of pressure by manometry, or combined with volume during rectal balloon distention. Pressure-volume (p-v) relationships provide a global index of rectal mechanical properties. However, balloon distention alone does not measure luminal radius or wall thickness, which are necessary to compute wall tension and stress respectively. It has been suggested that the elastic modulus, which is the linear slope of the stress-strain relationship, is a more accurate measure of wall stiffness. Also, measurements of compliance may not reflect differences in rectal diameter between subjects prior to inflation, and imaging is necessary to determine if, as has been suggested, rectal pressure-volume relationships are affected by extra-rectal structures. We have developed a technique to measure rectal stress:strain relationships in humans, by simultaneous magnetic resonance imaging (MRI) during rectal balloon distention. After a conditioning distention, a rectal balloon was distended with water from 0 to 400 ml in 50 ml steps, and imaged at each step with MRI. The fluid filled balloon was segmented from each volume, the phase-ordered binary volumes were transformed into a geometric characterization of the inflated rectal surface. Taken together with measurements of balloon pressure and of rectal wall thickness, this model of the rectal surface was used to calculate regional values of curvature, tension, strain, and stress for the rectum. In summary, this technique has the unique ability to non-invasively measure the rectal stress:strain relationship and also determine if rectal expansion is limited by extra-rectal structures. This functional information allows the direct clinical analysis of rectal motor function and offers the potential for characterizing abnormal mechanical properties of the rectal wall in disease.

Haider, Clifton R.; Manduca, Armando; Camp, Jon J.; Fletcher, Joel G.; Robb, Richard A.; Bharucha, Adil E.

2006-03-01

304

Effects of prostaglandin E and F receptor agonists in vivo on luteal function in ewes.  

PubMed

Loss of progesterone secretion at the end of the estrous cycle is via uterine PGF(2alpha) secretion; however, uterine PGF(2alpha) is not decreased during early pregnancy in ewes to prevent luteolysis. Instead the embryo imparts resistance to PGF(2alpha)-induced luteolysis, which is via the 2-fold increase in prostaglandins E(1) and E(2) (PGE(1), PGE(2); PGE) in the endometrium during early pregnancy. Chronic intrauterine infusion of PGE(1) or PGE(2) prevents spontaneous or an estradiol-17beta, IUD, or PGF(2alpha)-induced luteolysis. Four PGE receptor subtypes (EP(1), EP(2), EP(3), and EP(4)) and an FP receptor specific for PGF(2alpha) have been identified. The objective of this experiment was to determine the effects of EP(1), EP(2), EP(3), or FP receptor agonists in vivo on luteal mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone in ewes. Ewes received a single treatment of 17-phenyl-tri-Nor-PGE(2) (EP(1), EP(3)), butaprost (EP(2)), 19-(R)-OH-PGE(2) (EP(2)), sulprostone (EP(1), EP(3)), or PGF(2alpha) (FP) receptor agonists into the interstitial tissue of the ovarian vascular pedicle adjacent to the luteal-containing ovary. 17-Phenlyl-tri-Nor-PGE(2) had no effect (P> or =0.05) on any parameter analyzed. Butaprost and 19-(R)-OH-PGE(2) increased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. Both sulprostone and PGF(2alpha) decreased (P< or =0.05) mRNA for LH receptors, occupied and unoccupied LH receptors, and circulating progesterone. It is concluded that both EP(3) and FP receptors may be involved in luteolysis. In addition, EP(2) receptors may mediate prevention of luteolysis via regulation of luteal mRNA for LH receptors to prevent loss of occupied and unoccupied LH receptors and therefore to sustaining luteal function. PMID:20381634

Weems, Y S; Nett, T M; Rispoli, L A; Davis, T L; Johnson, D L; Uchima, T; Raney, A; Lennon, E; Harbert, T; Bowers, G; Tsutahara, N; Randel, R D; Weems, C W

2010-04-08

305

Beta-blockers, left and right ventricular function, and in-vivo calcium influx in muscular dystrophy cardiomyopathy.  

PubMed

Beta-blockers are used to treat acquired heart failure in adults, though their role in early muscular dystrophy cardiomyopathy is unclear. We treated 2 different dystrophic mouse models which have an associated cardiomyopathy (mdx: model for Duchenne Muscular Dystrophy, and Sgcd-/-: model for limb girdle muscular dystrophy type 2F) and wild type controls (C57 Bl10) with the beta blocker metoprolol or placebo for 8 weeks at an early stage in the development of the cardiomyopathy. Left and right ventricular function was assessed with cardiac magnetic resonance imaging (MRI) and in-vivo myocardial calcium influx with manganese enhanced MRI. In the mdx mice at baseline there was reduced stroke volume, cardiac index, and end-diastolic volume with preserved left ventricular ejection fraction. These abnormalities were no longer evident after treatment with beta-blockers. Right ventricular ejection fraction was reduced and right ventricular end-systolic volume increased in the mdx mice. With metoprolol there was an increase in right ventricular end-diastolic and end-systolic volumes. Left and right ventricular function was normal in the Sgcd-/- mice. Metroprolol had no significant effects on left and right ventricular function in these mice, though heart/body weight ratios increased after treatment. In-vivo myocardial calcium influx with MEMRI was significantly elevated in both models, though metoprolol had no significant effects on either. In conclusion, metoprolol treatment at an early stage in the development of cardiomyopathy has deleterious effects on right ventricular function in mdx mice and in both models no effect on increased in-vivo calcium influx. This suggests that clinical trials need to carefully monitor not just left ventricular function but also right ventricular function and other aspects of myocardial metabolism. PMID:23437355

Blain, Alison; Greally, Elizabeth; Laval, Steve; Blamire, Andrew; Straub, Volker; MacGowan, Guy A

2013-02-20

306

In vivo elemental analysis by counting neutron-induced gamma rays for medical and biological applications  

NASA Astrophysics Data System (ADS)

Non-invasive in vivo elemental analysis is a technique used to assess human body composition which is indicative of nutritional status and health condition. The in vivo measurement of the body's major elements is used for a variety of medical studies requiring the determination of the body's compartments (protein, fat, water, bone). Whole body gamma-ray counters, consisting of Nal(Tl) crystal detectors in a shielded room, are used for measuring in vivo the body's Ca, Cl, Na and P by delayed neutron activation analysis. Thermal neutrons from a moderated 238Pu-Be source are used for the measurement of total body nitrogen (and thus protein) and chlorine at low radiation exposure (0.80 mSv). The resulting high energy prompt gamma-rays from nitrogen (10.83 MeV) and chlorine (6.11 MeV) are detected simultaneously with the irradiation. Body fat (the main energy store) and fat distribution (which relates to risk for cardiovascular disease) are measured by detecting C and O in vivo through fast neutron inelastic scattering. A small sealed D-T neutron generator is used for the pulsed (4 - 8 KHz) production of fast neutrons. Carbon and oxygen are detected by counting the 4.44 and 6.13 MeV gamma-rays resulting from the inelastic scattering of the fast neutrons from the 12C and 16O nuclei, respectively. One use of this method is the systematic study of the mechanisms driving the age-associated depletion of the metabolizing, oxygen-consuming cellular compartment of the body. The understanding of this catabolism may suggest ways to maintain lean tissue and thus to preserve quality of life for the very old.

Kehayias, Joseph J.; Ma, Ruimei; Zhuang, Hong; Moore, Robert; Dowling, Lisa

1995-03-01

307

Semen analysis and sperm function testing  

PubMed Central

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm–zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.

Franken, Daniel R; Oehninger, Sergio

2012-01-01

308

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo.  

PubMed

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic-duodenal homeobox 1 (Pdx1)(+)/nestin(+) PPCs on islets. In vitro, co-culturing islets with Pdx1(+)/nestin(+) PPCs prolonged the former survival from 7 to 14 days. Furthermore, with high glucose (300.8 mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1(+)/nestin(+) PPCs for 3 days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2 weeks. Without Pdx1(+)/nestin(+) PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1(+)/nestin(+) PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo. PMID:22644623

Zhou, Shu-Yan; Zhang, Yu-Sen; Li, Qing; Zhang, Yi; Qi, Hui; Zhou, Han-Xin; Deng, Chun-Yan; Li, Fu-Rong

2012-05-29

309

In Vitro and In Vivo Studies of Single-Walled Carbon Nanohorns with Encapsulated Metallofullerenes and Exohedrally Functionalized Quantum Dots  

SciTech Connect

Single-walled carbon nanohorns (SWNHs) are new carbonaceous materials. In this paper, we report the first successful preparation of SWNHs encapsulating trimetallic nitride template endohedral metallofullerenes (TNT-EMFs). The resultant materials were functionalized by a high-speed vibration milling method and conjugated with CdSe/ZnS quantum dots (QDs). The successful encapsulation of TNT-EMFs and external functionalization with QDs provide a dual diagnostic platform for in vitro and in vivo biomedical applications of these new carbonaceous materials.

Zhang, Jianfei [Virginia Polytechnic Institute and State University (Virginia Tech); Ge, Jiechao [Virginia Polytechnic Institute and State University (Virginia Tech); Shultz, M.D. [Virginia Commonwealth University, Richland; Chung, Eunna [Virginia Polytechnic Institute and State University (Virginia Tech); Singh, Gurpreet [Virginia Polytechnic Institute and State University (Virginia Tech); Shu, Chunying [Virginia Polytechnic Institute and State University (Virginia Tech); Deck, Paul [Virginia Polytechnic Institute and State University (Virginia Tech); Fatouros, Panos [Virginia Commonwealth University, Richland; Henderson, Scott [Virginia Commonwealth University, Richland; Corwin, Frank [Virginia Commonwealth University, Richland; Geohegan, David B [ORNL; Rouleau, Christopher M [ORNL; More, Karren Leslie [ORNL; Rylander, Nichole M [Virginia Polytechnic Institute and State University (Virginia Tech); Rylander, Christopher [Virginia Polytechnic Institute and State University (Virginia Tech); Gibson, Harry W [Virginia Polytechnic Institute and State University (Virginia Tech); Dorn, Harry C [Virginia Polytechnic Institute and State University (Virginia Tech)

2010-07-01

310

Improving the in vivo persistence, distribution and function of cytotoxic T lymphocytes by inhibiting the tumor immunosuppressive microenvironment.  

PubMed

Adoptive cell transfer immunotherapy of malignant tumors has the problem of symbiosis between effector cells and tumor cells, a short in vivo residence time, and a poor killing efficiency of effector cells. Thus, releasing effector cells from the cancer immunosuppressive microenvironment and improving their effective time and functional status in vivo would seem to be ideal strategies for facilitating immunotherapy. Low-dose cyclophosphamide administration can effectively break immunotolerance by inhibiting regulatory T cells. In the present study, in order to verify whether the persistence, distribution and function of effector cells can be improved by inhibiting immunosuppressive microenvironment, low-dose cyclophosphamide was previously intraperitoneally injected into melanoma-bearing C57BL/6 mice, thereafter, CFSE-labeled cytotoxic T lymphocytes were transfused intravenously, and their effective time, distributive pattern, and killing efficiency in different groups were observed by measuring the fluorescence intensity and cell cycle of cytotoxic T lymphocytes distributed in various organs, in comparison with tumor growth. We found down-regulating Tregs in vivo can simultaneously reduce the levels of interleukin-10 and transforming growth factor-?. Migration and distribution of cytotoxic T lymphocytes in vivo was found to vary with time. Inhibition of immunotolerance can significantly improve the persistence, distribution, and function of cytotoxic T lymphocytes. Correspondingly, significantly higher secretion of perforin, granzyme B, IL-2, and IFN-? in tumor tissues with decreased tumor growth was seen in the cyclophosphamide injection group than in the control group. Our study may provide useful information on the cyclophosphamide-mediated mechanism for facilitating tumor immunotherapy by inhibiting the immunosuppressive tumor microenvironment. PMID:23659474

Xu, W; Cai, J; Li, S; Zhang, H; Han, J; Wen, M; Wen, J; Gao, F

2013-07-01

311

In vivo DNA expression of functional brome mosaic virus RNA replicons in Saccharomyces cerevisiae.  

PubMed Central

To facilitate manipulation of brome mosaic virus (BMV) RNA replicons in Saccharomyces cerevisiae and for yeast genetic analysis of BMV RNA replication, gene expression, and host interactions, we constructed DNA plasmids from which BMV RNA3 and RNA3 derivatives can be transcribed in vivo from the galactose-inducible yeast GAL1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. In galactose-induced yeast harboring such plasmids, expression of BMV RNA replication proteins 1a and 2a led to synthesis of negative-strand RNA3, amplification of positive-strand RNA3 to levels over 45-fold higher than those of DNA-derived RNA3 transcripts, and synthesis of the RNA3-encoded subgenomic mRNA for coat protein. Although the GAL1 promoter initiated transcription from multiple sites, 1a and 2a selectively amplified RNA3 with the authentic viral 5' end. As expected, reporter genes substituted for the 3'-proximal coat protein gene could not be translated directly from DNA-derived RNA3 transcripts, so their expression depended on 1a- and 2a-directed subgenomic mRNA synthesis. In yeast in which DNA transcription of B3CAT, an RNA3 derivative with the chloramphenicol acetyltransferase (CAT) gene replacing the coat gene, was induced, CAT activity remained near background levels in the absence of 1a and 2a but increased over 500,000-fold when 1a and 2a were expressed. Similarly, a plasmid encoding B3URA3, an RNA3 derivative with the yeast URA3 gene replacing the coat gene, conferred uracil-independent growth to ura3- yeast only after 1a and 2a expression and galactose induction. Once its 1a- and 2a-dependent replication was initiated, B3URA3 was maintained in dividing yeast as a free RNA replicon, even after repression of the GAL1 promoter or the loss of the B3URA3 cDNA plasmid. These findings should be useful for many experimental purposes.

Ishikawa, M; Janda, M; Krol, M A; Ahlquist, P

1997-01-01

312

Analysis of the Dynein-Dynactin Interaction In Vitro and In Vivo  

PubMed Central

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

King, Stephen J.; Brown, Christa L.; Maier, Kerstin C.; Quintyne, Nicholas J.; Schroer, Trina A.

2003-01-01

313

In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function.  

PubMed Central

To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo. Images Fig. 2 Fig. 5

Michelson, A D; Barnard, M R; Hechtman, H B; MacGregor, H; Connolly, R J; Loscalzo, J; Valeri, C R

1996-01-01

314

Functional analysis of inappropriate mealtime behaviors.  

PubMed Central

The purpose of the current investigation was to apply the functional analysis described by Iwata, Dorsey, Slifer, Bauman, and Richman (1982/1994) to the inappropriate mealtime behaviors of 15 children who had been referred to an intensive program for the assessment and treatment of severe feeding disorders. During Study 1, we conducted descriptive assessments of children and parents during meals. The results of Study 1 showed that parents used the following consequences for inappropriate mealtime behaviors: coaxing and reprimanding, allowing the child to periodically take a break from or avoid eating, and giving the child preferred food or toys following inappropriate behavior. The effects of these consequences were tested systematically in Study 2 when we conducted analogue functional analyses with the children. During alternating meals, one of the consequences typically used by parents consistently followed inappropriate child behavior. Results indicated that these consequences actually worsened behavior for 10 of the 15 children (67%). These results suggested that the analogue functional analysis described by Iwata et al. may be useful in identifying the environmental events that play a role in feeding disorders.

Piazza, Cathleen C; Fisher, Wayne W; Brown, Kimberly A; Shore, Bridget A; Patel, Meeta R; Katz, Richard M; Sevin, Bart M; Gulotta, Charles S; Blakely-Smith, Audrey

2003-01-01

315

Functional Analysis of Arabidopsis Sucrose Transporters  

SciTech Connect

Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter from companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.

John M. Ward

2009-03-31

316

Therapeutic nanomedicine based on dual-intelligent functionalized gold nanoparticles for cancer imaging and therapy in vivo.  

PubMed

A novel strategy to construct a therapeutic system based on functionalized AuNPs which can specifically respond to tumor microenvironment was reported. In the therapeutic system, doxorubicin was conjugated to AuNPs via thiol-Au bond by using a peptide substrate, CPLGLAGG, which can be specifically cleaved by the protease. In vivo study shows that after injection of the functionalized AuNPs to the tumor-bearing mice, the over-expressed protease of MMP-2 in tumor tissue and intracellular GSH can lead to the rapid release of the anti-tumor drug (doxorubicin) from the functionalized AuNPs to inhibit tumor growth and realize fluorescently imaging simultaneously. The functionalized AuNPs with tumor-triggered drug release property can further improve the efficacy and reduce side effects significantly. PMID:23932289

Chen, Wei-Hai; Xu, Xiao-Ding; Jia, Hui-Zhen; Lei, Qi; Luo, Guo-Feng; Cheng, Si-Xue; Zhuo, Ren-Xi; Zhang, Xian-Zheng

2013-08-09

317

Medical applications of in vivo neutron inelastic scattering and neutron activation analysis: Technical similarities to detection of explosives and contraband  

NASA Astrophysics Data System (ADS)

Nutritional status of patients can be evaluated by monitoring changes in elemental body composition. Fast neutron activation (for N and P) and neutron inelastic scattering (for C and O) are used in vivo to assess elements characteristic of specific body compartments. There are similarities between the body composition techniques and the detection of hidden explosives and narcotics. All samples have to be examined in depth and the ratio of elements provides a ``signature'' of the chemical of interest. The N/H and C/O ratios measure protein and fat content in the body. Similarly, a high C/O ratio is characteristic of narcotics and a low C/O together with a strong presence of N is a signature of some explosives. The available time for medical applications is about 20 min-compared to a few seconds for the detection of explosives-but the permitted radiation exposure is limited. In vivo neutron analysis is used to measure H, O, C, N, P, Na, Cl, and Ca for the study of the mechanisms of lean tissue depletion with aging and wasting diseases, and to investigate methods of preserving function and quality of life in the elderly. .

Kehayias, J. J.

2001-07-01

318

Ex vivo analysis of human memory B lymphocytes specific for a and B influenza hemagglutinin by polychromatic flow-cytometry.  

PubMed

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA(+) B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses. PMID:23976947

Bardelli, Monia; Alleri, Liliana; Angiolini, Francesca; Buricchi, Francesca; Tavarini, Simona; Sammicheli, Chiara; Nuti, Sandra; Degl'innocenti, Elena; Isnardi, Isabelle; Fragapane, Elena; Del Giudice, Giuseppe; Castellino, Flora; Galli, Grazia

2013-08-15

319

Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter FunctionsD?  

PubMed Central

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1–actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

Quintero-Monzon, Omar; Rodal, Avital A.; Strokopytov, Boris; Almo, Steven C.; Goode, Bruce L.

2005-01-01

320

3-D in vivo brain tumor geometry study by scaling analysis  

NASA Astrophysics Data System (ADS)

A new method, based on scaling analysis, is used to calculate fractal dimension and local roughness exponents to characterize in vivo 3-D tumor growth in the brain. Image acquisition was made according to the standard protocol used for brain radiotherapy and radiosurgery, i.e., axial, coronal and sagittal magnetic resonance T1-weighted images, and comprising the brain volume for image registration. Image segmentation was performed by the application of the k-means procedure upon contrasted images. We analyzed glioblastomas, astrocytomas, metastases and benign brain tumors. The results show significant variations of the parameters depending on the tumor stage and histological origin.

Torres Hoyos, F.; Martín-Landrove, M.

2012-02-01

321

Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin.  

PubMed

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content. (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim). PMID:23576407

Fereidouni, Farzad; Bader, Arjen N; Colonna, Anne; Gerritsen, Hans C

2013-04-11

322

PUREX Plant deactivation function analysis report  

SciTech Connect

The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and a function hierarchy chart that describe what needs to be performed to deactivate PUREX.

Lund, D.P.; PUREX Working Group

1995-09-01

323

FFTF Plant transition function analysis report  

SciTech Connect

The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and function hierarchy charts that describe what needs to be performed to deactivate FFTF.

Lund, D.P.; FFTF Working Group

1995-09-01

324

308 Building deactivation function analysis report  

SciTech Connect

The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and a function hierarchy chart that describes what needs to be performed to deactivate the 308 Building.

Lund, D.P.; 308 Building Working Group

1995-09-01

325

309 Building deactivation function analysis report  

SciTech Connect

The document contains the functions, function definitions, function interfaces, function interface definitions, Input Computer Automated Manufacturing Definition (IDEFO) diagrams, and a function hierarchy chart that describe what needs to be performed to deactivate the 309 Building.

Lund, D.P.; 309 Building Working Group

1995-09-01

326

Applying microscopy to the analysis of nuclear structure and function.  

PubMed

One of the ultimate goals of biological research is to understand mechanisms of cell function within living organisms. With this in mind, many sophisticated technologies that allow us to inspect macromolecular structure in exquisite detail have been developed. Although knowledge of structure derived from techniques such as X-ray crystallography and nuclear magnetic resonance is of vital importance, these approaches cannot reveal the remarkable complexity of molecular interactions that exists in vivo. With this in mind, this review focuses on the use of microscopy techniques to analyze cell structure and function. We describe the different basic microscopic methodologies and how the routine techniques are best applied to particular biological problems. We also emphasize the specific capabilities and uses of light and electron microscopy and highlight their individual advantages and disadvantages. For completion, we also comment on the alternative possibilities provided by a variety of advanced imaging technologies. We hope that this brief analysis of the undoubted power of microscopy techniques will be enough to stimulate a wider participation in this rapidly developing area of biological discovery. PMID:12606219

Iborra, Francisco; Cook, Peter R; Jackson, Dean A

2003-02-01

327

Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.  

PubMed Central

Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance.

Teixeira, M T; Siniossoglou, S; Podtelejnikov, S; Benichou, J C; Mann, M; Dujon, B; Hurt, E; Fabre, E

1997-01-01

328

Application of locked nucleic acids to improve aptamer in vivo stability and targeting function  

Microsoft Academic Search

Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure-activity rela- tionship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding.

Kathrin S. Schmidt; Sandra Borkowski; Jens Kurreck; Andrew W. Stephens; Rolf Bald; Maren Hecht; Matthias Friebe; Ludger Dinkelborg; Volker A. Erdmann

2004-01-01

329

Functionalized near-infrared quantum dots for in vivo tumor vasculature imaging  

Microsoft Academic Search

In this paper, we report the use of near-infrared (NIR)-emitting alloyed quantum dots (QDs) as efficient optical probes for high contrast in vivo imaging of tumors. Alloyed CdTe1 - xSex\\/CdS QDs were prepared in the non-aqueous phase using the hot colloidal synthesis approach. Water dispersion of the QDs were accomplished by their encapsulation within polyethyleneglycol (PEG)-grafted phospholipid micelles. For tumor-specific

Rui Hu; Ken-Tye Yong; Indrajit Roy; Hong Ding; Wing-Cheung Law; Hongxing Cai; Xihe Zhang; Lisa A. Vathy; Earl J. Bergey; Paras N. Prasad

2010-01-01

330

Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo.  

PubMed

Amnionless (AMN) and cubilin gene products appear to be essential functional subunits of an endocytic receptor called cubam. Mutation of either gene causes autosomal recessive Imerslund-Gräsbeck syndrome (I-GS, OMIM no. 261100) in humans, a disorder characterized by selective intestinal malabsorption of cobalamin (vitamin B12) and urinary loss of several specific low-molecular-weight proteins. Vital insight into the molecular pathology of I-GS has been obtained from studies of dogs with a similar syndrome. In this work, we show that I-GS segregates in a large canine kindred due to an in-frame deletion of 33 nucleotides in exon 10 of AMN. In a second, unrelated I-GS kindred, affected dogs exhibit a homozygous substitution in the AMN translation initiation codon. Studies in vivo demonstrated that both mutations abrogate AMN expression and block cubilin processing and targeting to the apical membrane. The essential features of AMN dysfunction observed in vivo are recapitulated in a heterologous cell-transfection system, thus validating the system for analysis of AMN-cubilin interactions. Characterization of canine AMN mutations that cause I-GS establishes the canine model as an ortholog of the human disorder well suited to studies of AMN function and coevolution with cubilin. PMID:15845892

He, Qianchuan; Madsen, Mette; Kilkenney, Adam; Gregory, Brittany; Christensen, Erik I; Vorum, Henrik; Hřjrup, Peter; Schäffer, Alejandro A; Kirkness, Ewen F; Tanner, Stephan M; de la Chapelle, Albert; Giger, Urs; Moestrup, Sřren K; Fyfe, John C

2005-04-21

331

Correlation energy functional from jellium surface analysis  

NASA Astrophysics Data System (ADS)

Using the wave-vector analysis of the jellium exchange-correlation surface energy, we show that the PBEint generalized gradient approximation (GGA) of Fabiano et al. [Phys. Rev. BPRBMDO1098-012110.1103/PhysRevB.82.113104 82, 113104 (2010)] is one of the most accurate density functionals for jellium surfaces, being able to describe both exchange and correlation parts of the surface energy, without error compensations. We show that the stabilized jellium model allows us to achieve a realistic description of the correlation surface energy of simple metals at any wave vector k. The PBEint correlation is then used to construct a meta-GGA correlation functional, modifying the one-electron self-correlation-free Tao-Perdew-Staroverov-Scuseria (TPSS) one. We find that this new functional (named JS) performs in agreement with fixed-node diffusion Monte Carlo estimates of the jellium surfaces, and is accurate for spherical atoms and ions of different spin-polarization and for Hooke’s atom for any value of the spring constant.

Constantin, Lucian A.; Chiodo, Letizia; Fabiano, Eduardo; Bodrenko, Igor; Sala, Fabio Della

2011-07-01

332

Spatial Analysis of Linguistic Data with GIS Functions  

Microsoft Academic Search

During the 1980s techniques for analysis of geographical patterns have been refined to the point that they may be applied to data from many fields. Quantitative spatial analysis and existing functions available in geographical information systems (GIS) enable computerized implementations of these spatial analysis methods. This paper describes the application of quantitative spatial analysis and GIS functions to analysis of

Jay Lee; William A. Kretzschmar Jr.

1993-01-01

333

Arginyltransferase is an ATP-Independent Self-Regulating Enzyme that Forms Distinct Functional Complexes In Vivo  

PubMed Central

Summary Posttranslational arginylation mediated by arginyltransferase (ATE1) plays an important role in cardiovascular development, cell motility and regulation of cytoskeleton and metabolic enzymes. This protein modification was discovered decades ago, however, the arginylation reaction and the functioning of ATE1 remained poorly understood due to the lack of good biochemical models. Here we report the development of an in vitro arginylation system, in which ATE1 function and molecular requirements can be tested using purified recombinant ATE1 isoforms supplemented with a controlled number of components. Our results show that arginylation reaction is a self-sufficient, ATP-independent process that can affect different sites in a polypeptide, and that arginyltransferases form different molecular complexes in vivo, associate with components of the translation machinery, and have distinct, partially overlapping subsets of substrates, suggesting that these enzymes play different physiological functions.

Wang, Junling; Han, Xuemei; Saha, Sougata; Xu, Tao; Rai, Reena; Zhang, Fangliang; Wolf, Yuri. I.; Wolfson, Alexey; Yates, John R.; Kashina, Anna

2010-01-01

334

The neurexin ligands, neuroligins and leucine-rich repeat transmembrane proteins, perform convergent and divergent synaptic functions in vivo  

PubMed Central

Synaptic cell adhesion molecules, including the neurexin ligands, neuroligins (NLs) and leucine-rich repeat transmembrane proteins (LRRTMs), are thought to organize synapse assembly and specify synapse function. To test the synaptic role of these molecules in vivo, we performed lentivirally mediated knockdown of NL3, LRRTM1, and LRRTM2 in CA1 pyramidal cells of WT and NL1 KO mice at postnatal day (P)0 (when synapses are forming) and P21 (when synapses are largely mature). P0 knockdown of NL3 in WT or NL1 KO neurons did not affect excitatory synaptic transmission, whereas P0 knockdown of LRRTM1 and LRRTM2 selectively reduced AMPA receptor-mediated synaptic currents. P0 triple knockdown of NL3 and both LRRTMs in NL1 KO mice yielded greater reductions in AMPA and NMDA receptor-mediated currents, suggesting functional redundancy between NLs and LRRTMs during early synapse development. In contrast, P21 knockdown of LRRTMs did not alter excitatory transmission, whereas NL manipulations supported a role for NL1 in maintaining NMDA receptor-mediated transmission. These results show that neurexin ligands in vivo form a dynamic synaptic cell adhesion network, with compensation between NLs and LRRTMs during early synapse development and functional divergence upon synapse maturation.

Soler-Llavina, Gilberto J.; Fuccillo, Marc V.; Ko, Jaewon; Sudhof, Thomas C.; Malenka, Robert C.

2011-01-01

335

Measuring stem cell frequency in epidermis: A quantitative in vivo functional assay for long-term repopulating cells  

NASA Astrophysics Data System (ADS)

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

Schneider, T. E.; Barland, C.; Alex, A. M.; Mancianti, M. L.; Lu, Y.; Cleaver, J. E.; Lawrence, H. J.; Ghadially, R.

2003-09-01

336

Fxr1 knockout mice show a striated muscle phenotype: implications for Fxr1p function in vivo  

Microsoft Academic Search

FXR1 is one of the two known homologues of FMR1. FXR1 shares a high degree\\u000a of sequence homology with FMR1 and also encodes two KH domains and an RGG\\u000a domain, conferring RNA-binding capabilities. In comparison with FMRP, very\\u000a little is known about the function of FXR1P in vivo. Mouse knockout (KO)\\u000a models exist for both Fmr1 and Fxr2. To study

Edwin J. Mientjes; Rob Willemsen; Laura L. Kirkpatrick; Ingeborg M. Nieuwenhuizen; Marianne Hoogeveen-Westerveld; Marcel Verweij; Surya Reis; Barbara Bardoni; Andre T. Hoogeveen; Ben A. Oostra; David L. Nelson

2004-01-01

337

In vivo wear and migration of highly cross-linked polyethylene cups a radiostereometry analysis study.  

PubMed

In 50 cemented hip arthroplasties, wear and migration of the polyethylene (PE) cups were measured with radiostereometric analysis for a period of 2 years. Twenty had a normal gamma-in-air-sterilized PE, another 20 had a PE sterilized with 30000 Gy followed by heat stabilization (Duration; Stryker Orthopaedics, Mahwah, NJ), and 10 had highly cross-linked PE cups irradiated with 100000 Gy (Crossfire; Stryker Orthopaedics). In the initial 2 months, head penetration (creep) was 63 microm on average for the 3 groups. From 2 to 24 months, the mean proximal head penetration (wear) was 156 microm for standard PE, 138 microm for stabilized PE (P = .45), and 23 microm for highly cross-linked PE (P < .001; analysis of variance). The low in vivo wear rate for highly cross-linked cups was not at the expense of higher migration or less favorable clinical outcome and looks promising. PMID:16124954

Röhrl, Stephan; Nivbrant, Bo; Mingguo, Li; Hewitt, Ben

2005-06-01

338

Epigenetic modulation of human breast cancer by metallofullerenol nanoparticles: in vivo treatment and in vitro analysis  

NASA Astrophysics Data System (ADS)

Multi-hydroxylated endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles possess the general physico-chemical characteristics of most nanoparticles. They also exhibit uniquely low toxicity and antineoplastic efficacy. In the current study, the molecular mechanisms and epigenetic characteristics of the antineoplastic action of these nanoparticles are explored. Human breast cancer MCF-7 and human umbilical vein endothelial ECV304 cell lines were used. Cell viability assay, cell hierarchical cluster analysis by cDNA microarray, semi-quantitative reverse transcription-polymerase chain reaction and Western blot analysis were conducted to investigate the changes in molecular and cellular signaling pathways caused by [Gd@C82(OH)22]n. The results demonstrated the high antitumor activity and low cytotoxicity of [Gd@C82(OH)22]n nanoparticles both in vivo and in vitro. Their possible anti-tumor mechanisms were also discussed. The present study may provide new insight into the mechanism of action of these nanoparticles.

Meng, Jie; Xing, Jianmin; Wang, Yingze; Lu, Juan; Zhao, Yuliang; Gao, Xueyun; Wang, Paul C.; Jia, Lee; Liang, Xingjie

2011-11-01

339

Functional Analysis of the Primate Shoulder  

PubMed Central

Studies of the shoulder girdle are in most cases restricted to morphological comparisons and rarely aim at elucidating function in a strictly biomechanical sense. To fill this gap, we investigated the basic functional conditions that occur in the shoulder joint and shoulder girdle of primates by means of mechanics. Because most of nonhuman primate locomotion is essentially quadrupedal walking—although on very variable substrates—our analysis started with quadrupedal postures. We identified the mechanical situation at the beginning, middle, and end of the load-bearing stance phase by constructing force parallelograms in the shoulder joint and the scapulo-thoracal connection. The resulting postulates concerning muscle activities are in agreement with electromyographical data in the literature. We determined the magnitude and directions of the internal forces and explored mechanically optimal shapes of proximal humerus, scapula, and clavicula using the Finite Element Method. Next we considered mechanical functions other than quadrupedal walking, such as suspension and brachiation. Quadrupedal walking entails muscle activities and joint forces that require a long scapula, the cranial margin of which has about the same length as the axillary margin. Loading of the hand in positions above the head and suspensory behaviors lead to force flows along the axillary margin and so necessitate a scapula with an extended axillary and a shorter cranial margin. In all cases, the facies glenoidalis is nearly normal to the calculated joint forces. In anterior view, terrestrial monkeys chose a direction of the ground reaction force requiring (moderate) activity of the abductors of the shoulder joint, whereas more arboreal monkeys prefer postures that necessitate activity of the adductors of the forelimb even when walking along branches. The same adducting and retracting muscles are recruited in various forms of suspension. As a mechanical consequence, the scapula is in a more frontal, rather than parasagittal, position on the thorax. In both forms of locomotion—quadrupedal walking and suspension—the compression-resistant clavicula contributes to keeping the shoulder complex distant from the rib cage. Future studies should consider the consequences for thorax shape. The morphological specializations of all Hominoidea match the functional requirements of suspensory behavior. The knowledge of mechanical functions allows an improved interpretation of fossils beyond morphological similarity.

Hohn, Bianca; Scherf, Heike; Schmidt, Manuela; Krause, Cornelia; Witzel, Ulrich

2010-01-01

340

Functional Analysis of the Primate Shoulder.  

PubMed

Studies of the shoulder girdle are in most cases restricted to morphological comparisons and rarely aim at elucidating function in a strictly biomechanical sense. To fill this gap, we investigated the basic functional conditions that occur in the shoulder joint and shoulder girdle of primates by means of mechanics. Because most of nonhuman primate locomotion is essentially quadrupedal walking-although on very variable substrates-our analysis started with quadrupedal postures. We identified the mechanical situation at the beginning, middle, and end of the load-bearing stance phase by constructing force parallelograms in the shoulder joint and the scapulo-thoracal connection. The resulting postulates concerning muscle activities are in agreement with electromyographical data in the literature. We determined the magnitude and directions of the internal forces and explored mechanically optimal shapes of proximal humerus, scapula, and clavicula using the Finite Element Method. Next we considered mechanical functions other than quadrupedal walking, such as suspension and brachiation. Quadrupedal walking entails muscle activities and joint forces that require a long scapula, the cranial margin of which has about the same length as the axillary margin. Loading of the hand in positions above the head and suspensory behaviors lead to force flows along the axillary margin and so necessitate a scapula with an extended axillary and a shorter cranial margin. In all cases, the facies glenoidalis is nearly normal to the calculated joint forces. In anterior view, terrestrial monkeys chose a direction of the ground reaction force requiring (moderate) activity of the abductors of the shoulder joint, whereas more arboreal monkeys prefer postures that necessitate activity of the adductors of the forelimb even when walking along branches. The same adducting and retracting muscles are recruited in various forms of suspension. As a mechanical consequence, the scapula is in a more frontal, rather than parasagittal, position on the thorax. In both forms of locomotion-quadrupedal walking and suspension-the compression-resistant clavicula contributes to keeping the shoulder complex distant from the rib cage. Future studies should consider the consequences for thorax shape. The morphological specializations of all Hominoidea match the functional requirements of suspensory behavior. The knowledge of mechanical functions allows an improved interpretation of fossils beyond morphological similarity. PMID:20495602

Preuschoft, Holger; Hohn, Bianca; Scherf, Heike; Schmidt, Manuela; Krause, Cornelia; Witzel, Ulrich

2010-04-13

341

Directed dimerization: an in vivo expression system for functional studies of type II phytochromes.  

PubMed

Type II phytochromes (phy) in Arabidopsis form homodimers and heterodimers, resulting in a diverse collection of light-stable red/far-red (R/FR) sensing photoreceptors. We describe an in vivo protein engineering system and its use in characterizing the activities of these molecules. Using a phyB null mutant background, singly and doubly transgenic plants were generated that express fusion proteins containing the phyB-phyE N-terminal photosensory regions (NB-NE PSRs), a nuclear localization sequence, and small yeast protein domains that mediate either homodimerization or heterodimerization. Activity of NB/NB homodimers but not monomeric NB subunits in control of seedling and adult plant responses to R light is demonstrated. Heterodimers of the NB sequence with the chromophoreless NB(C357S) sequence, which mimic phyB Pfr/Pr photo-heterodimers, mediate R sensitivity in leaves and petioles but not hypocotyls. Homodimerization of the NC, ND and NE sequences and directed heterodimerization of these photosensory regions with the NB region reveal form-specific R-induced activities for different type II phy dimers. The experimental approach developed here of directed assembly of defined protein dimer combinations in vivo may be applicable to other systems. PMID:23738620

Liu, Peng; Sharrock, Robert A

2013-08-03

342

Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation.  

PubMed

Near-infrared fluorophore (NIRF)-labeled imaging probes are becoming increasingly important in bio-molecular imaging applications, that is, in animal models for tumor imaging or inflammation studies. In this study we showed that the previously introduced chemical concept of 'Reverse Design' represents an efficient strategy for the generation of selective probes for cysteine proteases from chemically optimized protease inhibitors for investigations in proteomic lysates as well as for in vivo molecular imaging studies. The newly developed activity-based probe AW-091 was demonstrated to be highly selective for cathepsin S in vitro and proved useful in monitoring cysteine cathepsin activity in vivo, that is, in zymosan-induced mouse model of inflammation. AW-091 showed higher signal-to-background ratios at earlier time points than the commercially available polymer-based ProSense680 (VisEn Medical) and thus represents an efficient new tool for studying early proteolytic processes leading to various diseases, including inflammation, cancer, and rheumatoid arthritis. In addition, the fluorescent signal originating from the cleaved AW-091 was shown to be reduced by the administration of an anti-inflammatory drug, dexamethasone and by the cathepsin inhibitor E-64, providing a valuable system for the evaluation of small-molecule inhibitors of cathepsins. PMID:21130662

Cagli?, Dejan; Globisch, Anja; Kindermann, Maik; Lim, Ngee-Han; Jeske, Volker; Juretschke, Hans-Paul; Bartnik, Eckart; Weithmann, K Ulrich; Nagase, Hideaki; Turk, Boris; Wendt, K Ulrich

2010-10-19

343

Functional analysis of an arthritogenic synovial fibroblast  

PubMed Central

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.

Aidinis, Vassilis; Plows, David; Haralambous, Sylva; Armaka, Maria; Papadopoulos, Petros; Kanaki, Maria Zambia; Koczan, Dirk; Thiesen, Hans Juergen; Kollias, George

2003-01-01

344

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial ?? T cells is functional in vivo.  

PubMed

Intestinal intraepithelial lymphocytes carrying the ?? TCR (?? iIEL) are involved in the maintenance of epithelial integrity. ?? iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of ?? iIEL express the CD8?? homodimer. However, our knowledge about cognate ligands for most ?? TCR remains fragmentary and recent advances show that ?? T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of ?? iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor ? locus; H2B, histone 2B) reporter mice to identify ?? T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic ?? T cells, CD8??(+) ?? iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-? upon TCR triggering in vitro. Notably, in vivo blocking of the ?? TCR with specific mAb led to a decrease of basal calcium levels in CD8??(+) ?? iIEL. This suggests that the ?? TCR of CD8??(+) ?? iIEL is constantly being triggered and therefore functional in vivo. PMID:21108461

Malinarich, Frano H; Grabski, Elena; Worbs, Tim; Chennupati, Vijaykumar; Haas, Jan D; Schmitz, Susanne; Candia, Enzo; Quera, Rodrigo; Malissen, Bernard; Förster, Reinhold; Hermoso, Marcela; Prinz, Immo

2010-11-11

345

Regulation of memory CD4 T-cell pool size and function by natural killer T cells in vivo  

PubMed Central

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4+ T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naďve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-? expression by memory Th2 cells. These IFN-?–producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.

Iwamura, Chiaki; Shinoda, Kenta; Endo, Yusuke; Watanabe, Yukiko; Tumes, Damon John; Motohashi, Shinichiro; Kawahara, Kazuyoshi; Kinjo, Yuki; Nakayama, Toshinori

2012-01-01

346

Impact of nonnatural amino acid mutagenesis on the in vivo function and binding modes of a transcriptional activator.  

PubMed

Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biological networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the nonnatural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (S. cerevisiae). Functional studies of the pBpa-containing Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo cross-linking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Cross-linking with a range of pBpa-containing mutants revealed a Gal4 x Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be critical for the development of therapeutically useful small molecule modulators of these protein-protein interactions. PMID:19764747

Majmudar, Chinmay Y; Lee, Lori W; Lancia, Jody K; Nwokoye, Adaora; Wang, Qian; Wands, Amberlyn M; Wang, Lei; Mapp, Anna K

2009-10-14

347

Functional analysis of the nuclear LIM domain interactor NLI.  

PubMed Central

LIM homeodomain and LIM-only (LMO) transcription factors contain two tandemly arranged Zn2+-binding LIM domains capable of mediating protein-protein interactions. These factors have restricted patterns of expression, are found in invertebrates as well as vertebrates, and are required for cell type specification in a variety of developing tissues. A recently identified, widely expressed protein, NLI, binds with high affinity to the LIM domains of LIM homeodomain and LMO proteins in vitro and in vivo. In this study, a 38-amino-acid fragment of NLI was found to be sufficient for the association of NLI with nuclear LIM domains. In addition, NLI was shown to form high affinity homodimers through the amino-terminal 200 amino acids, but dimerization of NLI was not required for association with the LIM homeodomain protein Lmxl. Chemical cross-linking analysis revealed higher-order complexes containing multiple NLI molecules bound to Lmx1, indicating that dimerization of NLI does not interfere with LIM domain interactions. Additionally, NLI formed complexes with Lmx1 on the rat insulin I promoter and inhibited the LIM domain-dependent synergistic transcriptional activation by Lmx1 and the basic helix-loop-helix protein E47 from the rat insulin I minienhancer. These studies indicate that NLI contains at least two functionally independent domains and may serve as a negative regulator of synergistic transcriptional responses which require direct interaction via LIM domains. Thus, NLI may regulate the transcriptional activity of LIM homeodomain proteins by determining specific partner interactions.

Jurata, L W; Gill, G N

1997-01-01

348

A critical analysis of current in vitro and in vivo angiogenesis assays  

PubMed Central

The study of angiogenesis has grown exponentially over the past 40 years with the recognition that angiogenesis is essential for numerous pathologies and, more recently, with the advent of successful drugs to inhibit angiogenesis in tumours. The main problem with angiogenesis research remains the choice of appropriate assays to evaluate the efficacy of potential new drugs and to identify potential targets within the angiogenic process. This selection is made more complex by the recognition that heterogeneity occurs, not only within the endothelial cells themselves, but also within the specific microenvironment to be studied. Thus, it is essential to choose the assay conditions and cell types that most closely resemble the angiogenic disease being studied. This is especially important when aiming to translate data from in vitro to in vivo and from preclinical to the clinic. Here we critically review and highlight recent advances in the principle assays in common use including those for endothelial cell proliferation, migration, differentiation and co-culture with fibroblasts and mural cells in vitro, vessel outgrowth from organ cultures and in vivo assays such as chick chorioallantoic membrane (CAM), zebrafish, sponge implantation, corneal, dorsal air sac, chamber and tumour angiogenesis models. Finally, we briefly discuss the direction likely to be taken in future studies, which include the use of increasingly sophisticated imaging analysis systems for data acquisition.

Staton, Carolyn A; Reed, Malcolm W R; Brown, Nicola J

2009-01-01

349

In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model  

NASA Astrophysics Data System (ADS)

The rodent calvarial defect model is commonly used to investigate bone regeneration and wound healing. This study presents a micro-computed tomography (micro-CT) methodology for measuring the bone mineral content (BMC) in a rat calvarial defect and validates it by estimating its precision error. Two defect models were implemented. A single 6 mm diameter defect was created in 20 rats, which were imaged in vivo for longitudinal experiments. Three 5 mm diameter defects were created in three additional rats, which were repeatedly imaged ex vivo to determine precision. Four control rats and four rats treated with bone morphogenetic protein were imaged at 3, 6, 9 and 12 weeks post-surgery. Scan parameters were 80 kVp, 0.45 mA and 180 mAs. Images were reconstructed with an isotropic resolution of 45 µm. At 6 weeks, the BMC in control animals (4.37 ± 0.66 mg) was significantly lower (p < 0.05) than that in treated rats (11.29 ± 1.01 mg). Linear regression between the BMC and bone fractional area, from 20 rats, showed a strong correlation (r2 = 0.70, p < 0.0001), indicating that the BMC can be used, in place of previous destructive analysis techniques, to characterize bone growth. The high precision (2.5%) of the micro-CT methodology indicates its utility in detecting small BMC changes in animals.

Umoh, Joseph U.; Sampaio, Arthur V.; Welch, Ian; Pitelka, Vasek; Goldberg, Harvey A.; Underhill, T. Michael; Holdsworth, David W.

2009-04-01

350

In vivo analysis of human skin anisotropy by polarization-sensitive optical coherence tomography  

NASA Astrophysics Data System (ADS)

Skin anisotropy is an important issue for plastic surgeons and cosmetics science. Cleavage lines, such as Langer's lines and relaxed skin tension lines (RSTLs), have been proposed as keys to understanding skin anisotropy. Collagen, a dominant dermal structural protein, forms a fibrous structure believed to play an important role in skin anisotropy. There have been few reports, however, on the relationship between the orientation of collagen fiber and the direction of the cleavage line. Collagen fiber has birefringence, a property analyzable in skin in three dimensions by high-speed polarization-sensitive optical coherence tomography (PS-OCT). Here we used PS-OCT for an in vivo analysis of anisotropic changes in the dermal birefringence of mechanically deformed human skin. The dermal birefringence of the forehead increased significantly when the skin was shrunk perpendicular to the RSTL and increased significantly when the skin was shrunk parallel to the RSTL. En-face images of dermal birefringence revealed that both shrinking perpendicular to and stretching in parallel to the RSTL promoted the formation of a macro rope-like collagen structure. Moreover, the birefringent change under shrinking conditions perpendicular to the RSTL showed negative correlation to Ra, a skin roughness parameter. These results suggest that PS-OCT enables the in vivo evaluation of skin anisotropy.

Sakai, Shingo; Yamanari, Masahiro; Lim, Yiheng; Makita, Shuichi; Nakagawa, Noriaki; Yasuno, Yoshiaki

2011-02-01

351

In Vivo Analysis of Growth Hormone Receptor Signaling Domains and Their Associated Transcripts  

PubMed Central

The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR?/? mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.

Rowland, Jennifer E.; Lichanska, Agnieszka M.; Kerr, Linda M.; White, Mary; d'Aniello, Elisabetta M.; Maher, Sheryl L.; Brown, Richard; Teasdale, Rohan D.; Noakes, Peter G.; Waters, Michael J.

2005-01-01

352

Delta-catenin is required for the maintenance of neural structure and function in mature cortex in vivo.  

PubMed

Delta-catenin is a brain-specific member of the adherens junction complex that localizes to the postsynaptic and dendritic compartments. This protein is likely critical for normal cognitive function; its hemizygous loss is linked to the severe mental retardation syndrome Cri-du-Chat and it directly interacts with presenilin-1 (PS1), the protein most frequently mutated in familial Alzheimer's disease. Here we examine dendritic structure and cortical function in vivo in mice lacking delta-catenin. We find that in cerebral cortex of 5-week-old mice, dendritic complexity, spine density, and cortical responsiveness are similar between mutant and littermate controls; thereafter, mutant mice experience progressive dendritic retraction, a reduction in spine density and stability, and concomitant reductions in cortical responsiveness. Our results indicate that delta-catenin regulates the maintenance of dendrites and dendritic spines in mature cortex but does not appear to be necessary for the initial establishment of these structures during development. PMID:19914181

Matter, Cheryl; Pribadi, Mochtar; Liu, Xin; Trachtenberg, Joshua T

2009-11-12

353

In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene  

PubMed Central

Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1?,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

Nakanishi, Tomoko; Saito, Rumiko; Taniguchi, Makoto; Oda, Haruka; Soma, Atsumi; Yasunaga, Mayu; Yamane, Mariko; Sato, Kenzo

2013-01-01

354

Chemical analysis in vivo and in vitro by Raman spectroscopy - from single cells to humans  

PubMed Central

Summary The gold standard for clinical diagnostics of tissues is immunofluorescence staining. Toxicity of many fluorescent dyes precludes their application in vivo. Raman spectroscopy, a chemically specific, label-free diagnostic technique, is rapidly gaining in acceptance as a powerful alternative. It has the ability to probe the chemical composition of biological materials in a nondestructive and mostly non-perturbing manner. We review the most recent developments in Raman spectroscopy in the life sciences, detailing advances in technology that have improved the ability to screen for diseases. Its role in the monitoring of biological function and mapping the intracellular chemical microenvironment will be discussed. Applications including endoscopy, surface-enhanced Raman scattering (SERS), and coherent Raman scattering (CRS) will be reviewed.

Wachsmann-Hogiu, Sebastian; Weeks, Tyler

2009-01-01

355

REVIEW ARTICLE: Transfer function analysis of radiographic imaging systems  

Microsoft Academic Search

The following subjects are considered: principles of transfer function analysis of imaging systems (including: linear, shift-invariant imaging systems; Fourier analysis; transfer function analysis); applications to radiographic imaging systems (including: system linearisation; the object; geometrical unsharpness (GU); screen-film system unsharpness (SFSU); combined effects of GU and SFSU; other unsharpness effects; noise analysis).

C. E. Metz; K. Doi

1979-01-01

356

Analysis of 3D motion of in-vivo pacemaker leads  

NASA Astrophysics Data System (ADS)

In vivo analyses of pacemaker lead motion during the cardiac cycle have become important due to incidences of failure of some of the components. For the calculation and evaluation of in vivo stresses in pacemaker leads, the 3D motion of the lead must be determined. To accomplish this, we have developed a technique for calculation of the overall and relative 3D position, and thereby the 3D motion, of in vivo pacemaker leads through the cardiac cycle.Biplane image sequences of patients with pacemakers were acquired for at least two cardiac cycles. After the patient acquisitions, biplane images of a calibration phantom were obtained. The biplane imaging geometries were calculated from the images of the calibration phantom. Points on the electrodes and the lead centerlines were indicated manually in all acquired images. The indicated points along the leads were then fit using a cubic spline. In each projection, the cumulative arclength along the centerlines in two temporally adjacent images was used to identify corresponding points along the centerlines. To overcome the non-synchronicity of the biplane image acquisition, temporal interpolation was performed using these corresponding points based on a linear scheme. For each time point, corresponding points along the lead centerlines in the pairs of biplane images were identified using epipolar lines. The 3D lead centerlines were calculated from the calculated imaging geometries and the corresponding image points along the lead centerlines. From these data, 3D lead motion and the variations of the lead position with time were calculated and evaluated throughout the cardiac cycle. The reproducibility of the indicated lead centerlines was approximately 0.3 mm. The precision of the calculated rotation matrix and translation vector defining image geometry were approximately 2 mm. 3D positions were reproducible to within 2 mm. Relative positional errors were less than 0.3 mm. Lead motion correlated strongly with phases of the cardiac cycle. Our results indicate that complex motions of in vivo pacemaker leads can be precisely determined. Thus, we believe that this technique will provide precise 3D motion and shapes on which to base subsequent stress analysis of pacemaker lead components.

Hoffmann, Kenneth R.; Williams, Benjamin B.; Esthappan, Jacqueline; Chen, Shiuh-Yung J.; Fiebich, Martin; Carroll, John D.; Harauchi, Hajime; Doerr, Vince; Kay, G. Neal; Eberhardt, Allen; Overland, Mary

1997-04-01

357

In vivo biological responses to silk proteins functionalized with bone sialoprotein.  

PubMed

Recombinant 6mer?+?BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer?+?BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer?+?BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications. PMID:23359587

Gomes, Sílvia; Gallego-Llamas, Jabier; Leonor, Isabel B; Mano, Joăo F; Reis, Rui L; Kaplan, David L

2013-01-28

358

In vivo functional photoacoustic tomography of traumatic brain injury in rats  

NASA Astrophysics Data System (ADS)

In this study, we demonstrate the potential of photoacoustic tomography for the study of traumatic brain injury (TBI) in rats in vivo. Based on spectroscopic photoacoustic tomography that can detect the absorption rates of oxy- and deoxy-hemoglobins, the blood oxygen saturation and total blood volume in TBI rat brains were visualized. Reproducible cerebral trauma was induced using a fluid percussion TBI device. The time courses of the hemodynamic response following the trauma initiation were imaged with multi-wavelength photoacoustic tomography with bandwidth-limited spatial resolution through the intact skin and skull. In the pilot set of experiments, trauma induced hematomas and blood oxygen saturation level changes were detected, a finding consistent with the known physiological responses to TBI. This new imaging method will be useful for future studies on TBI-related metabolic activities and the effects of therapeutic agents.

Oh, Jung-Taek; Song, Kwang-Hyun; Li, Meng-Lin; Stoica, George; Wang, Lihong V.

2006-03-01

359

Lipopolysaccharide enhances Fc?R-dependent functions in vivo through CD11b/CD18 up-regulation  

PubMed Central

Fc receptors for immunoglobulin G (IgG) (Fc?R) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases Fc?R-dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-? (TNF-?) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fc?-dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fc?-receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting Fc?R expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of Fc?R functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and Fc?R could be critical for the modulation of effector functions during an inflammatory response.

Rubel, C; Miliani De Marval, P; Vermeulen, M; Isturiz, M A; Palermo, M S

1999-01-01

360

Truncated Moment Analysis of Nucleon Structure Functions  

Microsoft Academic Search

The understanding of quark-hadron duality in nucleon structure functions (namely, the similarity between the scaling and resonance averaged functions) within QCD is currently incomplete. While moments of structure functions can be analyzed within the operator product expansion in terms of leading and higher twist contributions, the description of duality as a function of Bjorken x requires phenomenological models. We employ

Ales Psaker; Eric Christy; Cynthia Keppel; Wally Melnitchouk

2007-01-01

361

The feasibility of accelerator-based in vivo neutron activation analysis of nitrogen.  

PubMed

The feasibility of accelerator-based in vivo neutron activation analysis of nitrogen has been investigated. It was found that a moderated neutron flux from approximately 10 microA of 2.5 MeV protons on a 9Be target performed as well as, and possibly slightly better than the existing isotope-based approach in terms of net counts per unit subject dose. Such a system may be an attractive alternative to the widespread use of (238,239)Pu/Be or 252Cf neutron sources, since there is more flexibility in the energy spectrum generated by accelerator-based neutron sources. From a radiation safety standpoint, accelerators have the advantage in that they only produce radiation when in operation. Furthermore, an accelerator beam can be pulsed, to reduce background detected in the prompt-gamma measurement, and such a device has a wide range of additional biological and medical applications. PMID:11761098

O'Meara, J M; Blackburn, B W; Chichester, D L; Gierga, D P; Yanch, J C

2001-12-01

362

Keratoglobus and posterior subcapsular cataract: surgical considerations and in vivo microstructural analysis.  

PubMed

We report sporadic, bilateral keratoglobus associated with posterior subcapsular cataract in a 43-year-old man. Slitlamp biomicroscopy showed symmetric arcus senilis-like deposits, a polygonal appearance resembling crocodile shagreen, an unusual endothelial appearance, and posterior subcapsular cataract. Orbscan II pachymetry maps (Bausch & Lomb) demonstrated bilateral diffuse corneal thinning (359.53 microm +/- 21.15 [SD] in the right eye and 379.61 +/- 11.49 microm in the left eye). These thickness values were confirmed by ultrasound pachymetry. In vivo confocal microscopy showed multiple criss-crossing dark lines and no identifiable cellular elements within the stroma. There were mild to moderate, guttata-like endothelial changes surrounded by pleomorphic cells. Phacoemulsification was performed in the left eye after careful consideration of the presenting features and modification of the surgical technique. Minimal structural alteration was observed during microstructural analysis 7 months after surgery. The endothelial morphology postoperatively was similar to that at baseline. PMID:14967295

Ku, Judy Y F; Grupcheva, Christina N; Fisk, Michael J; McGhee, Charles N J

2004-01-01

363

Correlation, functional analysis and optical pattern recognition  

SciTech Connect

Correlation integrals have played a central role in optical pattern recognition. The success of correlation, however, has been limited. What is needed is a mathematical operation more complex than correlation. Suitably complex operations are the functionals defined on the Hilbert space of Lebesgue square integrable functions. Correlation is a linear functional of a parameter. In this paper, we develop a representation of functionals in terms of inner products or equivalently correlation functions. We also discuss the role of functionals in neutral networks. Having established a broad relation of correlation to pattern recognition, we discuss the computation of correlation functions using acousto-optics.

Dickey, F.M.; Lee, M.L.; Stalker, K.T.

1994-03-01

364

Downregulation of the Antigen Presenting Cell Function(s) of Pulmonary Dendritic Cells In Vivo by Resident Alveolar Macrophages  

Microsoft Academic Search

Sllnllnal~ Class II major histocompatibility complex (Ia)-bearing dendritic cells (DC) from airway epithelium and lung parenchyma express low-moderate antigen presenting cell (APC) activity when freshly isolated. However, this function is markedly upregulated during overnight culture in a manner analogous to epidermal Langerhans cells. The in vitro \\

Patrick G. Holt; Jane Oliver; Natalie Bilyk; Christine McMenamin; Paul G. McMenamin; Georg Kraal

1993-01-01

365

Structure-function analysis of Frizzleds.  

PubMed

Frizzleds, cell surface receptors that mediate the actions of Wnt ligands on early development, are heptahelical (based upon hydropathy analysis) and couple to heterotrimeric G proteins. The primary structure of all ten mammalian Frizzleds display many landmarks observed in virtually all G protein-coupled receptors, including an exofacial N-terminus that is N-glycosylated, the presence of seven hydrophobic transmembrane segments predicted to form alpha-helixes, and three intracellular loops as well as a cytoplasmic, C-terminal tail that harbor suspected sites for protein phosphorylation. Prediction of the G proteins to which Frizzleds mediate signaling based upon a bioinformatic analysis of the primary sequence of the intracellular domains are in good agreement with functional screens in Drosophila, zebrafish, and mouse models of development, e.g., predicting Frizzled-1 to interact with members of the Gi/Go protein family. Likewise various Wnt signaling pathways are sensitive to treatment with pertussis toxin and knock-down of specific G protein alpha-subunits. Homology among the sequences encoding the cytoplasmic domains of human Frizzleds is high and the various Frizzleds can be segregated into subsets predicted to share some common downstream signaling elements. Among different species, homologies can reveal conservation of signaling to cognate G protein partners. Additionally, cytoplasmic domains of the prototypic beta2-adrenergic receptor can be substituted with those from either Frizzled-1 or Frizzled-2 to create chimeric receptors that are activated by beta-adrenergic agonists, yet signal with high fidelity to the Wnt/beta-catenin and Wnt/Ca2+, cyclic GMP pathways, respectively, regulating key aspects of early development. The nature of Frizzled-based signaling complexes, their temporal assembly, and spatial distribution via scaffold protein remains to be elucidated, as does whether or not these Wnt receptors display agonist-induced desensitization, internalization, and re-cycling to the cell membrane. PMID:16480852

Wang, Hsien-yu; Liu, Tong; Malbon, Craig C

2006-02-09

366

Structured Analysis/Design - LSA Tank 301, Functional Requirements Identification, Subtask 301.2.3, Functional Requirements Risk Analysis.  

National Technical Information Service (NTIS)

This report consolidates the Structured Analysis and Structured Design for the Logistic Support Analysis (LSA) Tasks. Included are the Data Flow Diagrams, (DFDs) for LSA Subtask 301.2.3, 'Functional Requirements Risk Analysis', and the corresponding descr...

R. Duclos N. Shepherd

1990-01-01

367

Geometric modeling, functional parameter calculation, and visualization of the in-vivo distended rectal wall  

Microsoft Academic Search

The rectum can distend to accommodate stool, and contracts in response to distention during defecation. Rectal motor dysfunctions are implicated in the pathophysiology of functional defecation disorders and fecal incontinence. These rectal motor functions can be studied by intra-luminal measurements of pressure by manometry, or combined with volume during rectal balloon distention. Pressure-volume (p-v) relationships provide a global index of

Clifton R. Haider; Armando Manduca; Jon J. Camp; Joel G. Fletcher; Richard A. Robb; Adil E. Bharucha

2006-01-01

368

Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-D-glucose gold nanoparticles in vivo.  

PubMed

We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-D-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the (18)F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning. PMID:22922305

Roa, Wilson; Xiong, Yeping; Chen, Jie; Yang, Xiaoyan; Song, Kun; Yang, Xiaohong; Kong, Beihua; Wilson, John; Xing, James Z

2012-08-24

369

Pharmacokinetic and toxicological evaluation of multi-functional thiol-6-fluoro-6-deoxy-d-glucose gold nanoparticles in vivo  

NASA Astrophysics Data System (ADS)

We synthesized a novel, multi-functional, radiosensitizing agent by covalently linking 6-fluoro-6-deoxy-d-glucose (6-FDG) to gold nanoparticles (6-FDG-GNPs) via a thiol functional group. We then assessed the bio-distribution and pharmacokinetic properties of 6-FDG-GNPs in vivo using a murine model. At 2 h, following intravenous injection of 6-FDG-GNPs into the murine model, approximately 30% of the 6-FDG-GNPs were distributed to three major organs: the liver, the spleen and the kidney. PEGylation of the 6-FDG-GNPs was found to significantly improve the bio-distribution of 6-FDG-GNPs by avoiding unintentional uptake into these organs, while simultaneously doubling the cellular uptake of GNPs in implanted breast MCF-7 adenocarcinoma. When combined with radiation, PEG-6-FDG-GNPs were found to increase the apoptosis of the MCF-7 breast adenocarinoma cells by radiation both in vitro and in vivo. Pharmacokinetic data indicate that GNPs reach their maximal concentrations at a time window of two to four hours post-injection, during which optimal radiation efficiency can be achieved. PEG-6-FDG-GNPs are thus novel nanoparticles that preferentially accumulate in targeted cancer cells where they act as potent radiosensitizing agents. Future research will aim to substitute the 18F atom into the 6-FDG molecule so that the PEG-6-FDG-GNPs can also function as radiotracers for use in positron emission tomography scanning to aid cancer diagnosis and image guided radiation therapy planning.

Roa, Wilson; Xiong, Yeping; Chen, Jie; Yang, Xiaoyan; Song, Kun; Yang, Xiaohong; Kong, Beihua; Wilson, John; Xing, James Z.

2012-09-01

370

Radiofrequency time-domain EPR imaging: instrumentation development and recent results in functional physiological in vivo imaging  

NASA Astrophysics Data System (ADS)

Electron Paramagnetic Resonance is an emerging technique finding applications in functional physiological imaging. Traditionally EPR imaging developed as a CW (continuous wave) technique involving the measurement of free radical distribution in vivo using constant frequency and field-sweep modality almost identical to the early developments of MRI. As in CT and PET this involved the generation of projections in presence of gradients and the reconstruction of images via filtered back-projection. The large line-width and the concomitant short relaxation times posed a serious challenge for the development of time-domain methods akin to modern pulsed NMR & MRI. With the recent availability of narrow line stable non-toxic radicals based on triarylmethyl (TAM), ultra fast data acquisition systems (signal digitizer and summer), very fast electronic switches and low-noise amplifiers, we have developed time-domain imaging schemes in EPR operating in the radiofrequency region Using a novel pure-phase encoding scheme, we are able to generate 2 and 3 dimensional spatial images and spectral-spatial images that adds an additional functional dimension to these images. The special space-encoding scheme with fast gradient ramping allow rapid in vivo imaging of small animals with superior spatial and functional information with good temporal resolution that can provide valuable physiological and pharmacokinetic insight. Our main thrust has been in the investigation of tumor hypoxia and tumor reoxygenation for the purpose of minimizing the radiation dose for maximum tumor cell killing. These and some of the allied imaging methods, and results from tumor investigation will be presented.

Subramanian, Sankaran; Devasahayam, Nallathamby; Krishna, M. C.

2007-03-01

371

Empirical Orthogonal Function Analysis of Hawaiian Rainfall.  

NASA Astrophysics Data System (ADS)

Empirical orthogonal function analysis was applied to monthly mean rainfall data at 63 stations in Hawaii encompassing a 37-year period. Major rainfall patterns in order to importance (E1-E3) proved to be trade wind, southwest wind and convective rainfall on an annual basis; trade wind, southwest wind and frontal rainfall during winter, spring and fall seasons; and trade wind, tropical disturbance and convective rainfall during summer. Trade wind rainfall (E1) explains most rainfall variance in summer and least variance in winter. Spectral analyses of the time-dependent coefficients for eigenvectors E1-E5 show annual, semi-annual, three-forths year, and 2-2 1/2 year cycles. No spectral peaks relating to the 11- and 22-year sunspot cycles were found. Composite rainfall maps for wet and dry winter and summer half-years indicate the contributions that specific eigenvector patterns make to these anomalies. Comparisons between Hawaiian rainfall and E1 Nińos reveal that most (not all) E1 Nińo winters in Hawaii are dry. Lack of trade wind rainfall is the primary cause.

Lyons, Steven W.

1982-11-01

372

In vivo and in vitro function of human UDP-galactose 4?-epimerase variants  

PubMed Central

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4?-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4?-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4?-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three ?-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.

McCorvie, Thomas J.; Wasilenko, Jamie; Liu, Ying; Fridovich-Keil, Judith L.; Timson, David J.

2011-01-01

373

In vivo functional chronic imaging of a small animal model using optical-resolution photoacoustic microscopy  

PubMed Central

Optical-resolution photoacoustic microscopy (OR-PAM) has been validated as a valuable tool for label-free volumetric microvascular imaging. More importantly, the advantages of noninvasiveness and measurement consistency suggest the use of OR-PAM for chronic imaging of intact microcirculation. Here, such chronic imaging is demonstrated for the first time by monitoring the healing process of laser-induced microvascular lesions in a small animal model in vivo. The central part of a 1 mm by 1 mm region in a nude mouse ear was treated under a continuous-wave laser to create a microvascular lesion for chronic study. The region of interest was imaged before the laser treatment, immediately after the treatment, and throughout the healing process using both the authors’ OR-PAM system and a commercial transmission-mode optical microscope. Three-dimensional microvascular morphology and blood oxygenation information were imaged simultaneously at capillary-level resolution. Transmission-mode optical microscopic images were acquired for comparison. OR-PAM has potential important applications in microcirculatory physiology or pathophysiology, tumor angiogenesis, laser microsurgery, and neuroscience.

Hu, Song; Maslov, Konstantin; Wang, Lihong V.

2009-01-01

374

Effects of ex vivo ?-Tocopherol on Airway Macrophage Function in Healthy and Mild Allergic Asthmatics.  

PubMed

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate ?-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor. PMID:23689260

Geiser, Marianne; Lay, John C; Bennett, William D; Zhou, Haibo; Wang, Xiaoyan; Peden, David B; Alexis, Neil E

2013-05-08

375

Failure analysis of two Ti-alloy total hip arthroplasty femoral stems fractured in vivo.  

PubMed

Failure of total hip arthroplasty femoral stems is a serious clinical complication. Even modern metal alloys and designs sometimes suffer such incidents. The reported study aimed at the investigation of the reasons leading the in vivo fracture of two Ti6Al4V femoral stems. Stems were retrieved during revision surgery approximately 2 years postoperatively. Examination and analysis included XRF spectrometry for identification of chemical composition, macroscopic examination and topographical measurements, SEM study of fracture surfaces, study of alloy microstructure by optical microscopy, and finally measurement of mechanical properties by means of tensile testing conducted on alloy samples machined from the stems themselves. Macroscopic examination and measurements showed close topographical similarity between the two fractures. XRF spectrometry, tensile testing, and microstructure analysis identified the alloy as a typical Ti6Al4V surgical titanium alloy. During SEM analysis the fracture surfaces exhibited characteristic fatigue striations tidally running on the cross sections, which were considered as the weakest ones regarding toward geometry and stress concentration. Fracture in both stems occurred due to fatigue along these cross sections. PMID:12808587

Magnissalis, E A; Zinelis, S; Karachalios, Th; Hartofilakidis, G

2003-07-15

376

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC)  

Microsoft Academic Search

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability\\u000a in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic\\u000a T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably\\u000a expand antigen specific

Malarvizhi Durai; Christine Krueger; Zhaohui Ye; Linzhao Cheng; Andreas Mackensen; Mathias Oelke; Jonathan P. Schneck

2009-01-01

377

Gene array analysis of the effects of chronic adrenocorticotropic hormone in vivo on immature rat adrenal glands  

Microsoft Academic Search

Development of a mature adrenocortical phenotype is a critical event in the transition of mammals from fetal to postnatal life. We previously reported that the functional maturation of the adrenal glands of newborn rats is accelerated by adrenocorticotropic hormone (ACTH). We report here that chronic exposure of neonatal\\/juvenile rat pups to ACTH in vivo results in significant changes in expression

Julie J. Lee; Eric P. Widmaier

2005-01-01

378

Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo  

Microsoft Academic Search

BACKGROUND: Acute enteritis caused by Salmonella is a public health concern. Salmonella infection is also known to increase the risk of inflammatory bowel diseases and cancer. Therefore, it is important to understand how Salmonella works in targeting eukaryotic pathways in intestinal infection. However, the global physiological function of Salmonella typhimurium in intestinal mucosa in vivo is unclear. In this study,

Xingyin Liu; Rong Lu; Yinglin Xia; Jun Sun

2010-01-01

379

Association of Marginal Folate Depletion with Increased Human Chromosomal Damage In vivo: Demonstration by Analysis of Micronucleated Erythrocytes.  

National Technical Information Service (NTIS)

Recent studies have demonstrated that in the absence of spleen function, frequencies of micronuclei (Howell-Jolly bodies) in peripheral rbcs can be used to measure in vivo cytogenetic damage. Among 20 subjects studied 6 months after splenectomy, 1 had a f...

R. B. Everson C. M. Wehr G. L. Erexson J. T. MacGregor

1988-01-01

380

ASSOCIATION OF MARGINAL FOLATE DEPLETION WITH INCREASED HUMAN CHROMOSOMAL DAMAGE IN VIVO: DEMONSTRATION BY ANALYSIS OF MICRONUCLEATED ERYTHROCYTES  

EPA Science Inventory

Recent studies have demonstrated that in the absence of spleen function, frequencies of micronuclei (Howell-Jolly bodies) in peripheral rbcs can be used to measure in vivo cytogenetic damage. mong 20 subjects studied 6 months after splenectomy 1 had a frequency of micronucleated ...

381

Mood states, sympathetic activity, and in vivo beta-adrenergic receptor function in a normal population.  

PubMed

The purpose of this study was to examine the relationship between mood states and beta-adrenergic receptor function in a normal population. We also examined if sympathetic nervous system activity is related to mood states or beta-adrenergic receptor function. Sixty-two participants aged 25-50 years were enrolled in this study. Mood states were assessed using the Profile of Mood States (POMS). Beta-adrenergic receptor function was determined using the chronotropic 25 dose isoproterenol infusion test. Level of sympathetic nervous system activity was estimated from 24-hr urine norepinephrine excretion. Higher tension-anxiety, depression-dejection, and anger-hostility were related to decreased beta-adrenergic receptor sensitivity (i.e., higher chronotropic 25 dose values), but tension-anxiety was the only remaining independent predictor of beta-adrenergic receptor function after controlling for age, gender, ethnicity, and body mass index (BMI). Urinary norepinephrine excretion was unrelated to either mood states or beta-adrenergic receptor function. These findings replicate previous reports that anxiety is related to decreased (i.e., desensitized) beta-adrenergic receptor sensitivity, even after controlling for age, gender, ethnicity, and body mass index. PMID:17583588

Yu, Bum-Hee; Kang, Eun-Ho; Ziegler, Michael G; Mills, Paul J; Dimsdale, Joel E

2008-01-01

382

Photoinactivation of functional photosystem II and D1-protein synthesis in vivo are independent of the modulation of the photosynthetic apparatus by growth irradiance  

Microsoft Academic Search

To investigate whether the in-vivo photoinhibition of photosystem II (PSII) function by excess light is an intrinsic property of PSII, the maximal photochemical efficiency of PSII (Fv\\/Fm) and the content of functional PSII (measured by repetitive flash yield of oxygen evolution) were determined in leaves of pea (Pisum sativum L.), grown in 50 (low light), 250 (medium light), and 650

Youn-Il Park; Jan M. Anderson; Wah Soon Chow

1996-01-01

383

In vivo investigation of luteal function in dogs: Effects of cabergoline, a dopamine agonist, and prolactin on progesterone secretion during mid-pregnancy and diestrus  

Microsoft Academic Search

The role of prolactin on luteal function in dogs was investigated in vivo. The function of prolactin in mid-luteal phase was compared in pregnant and nonpregnant dogs. A dopamine agonist, cabergoline, known for its prolactin secretion inhibitory effects, was injected subcutaneously at a dose of 5 ?g\\/kg body weight in five pregnant and five nonpregnant Beagle bitches. Mean plasma prolactin

K. Onclin; J. P. Verstegen

1997-01-01

384

Toll-like receptor 3 regulates cord blood-derived endothelial cell function in vitro and in vivo.  

PubMed

Circulating endothelial progenitor cells (cEPC) are capable of homing to neovascularisation sites, in which they proliferate and differentiate into endothelial cells. Transplantation of cEPC-derived cells, in particular those isolated from umbilical cord blood (UCB), has emerged as a promising approach in the treatment of cardio-vascular diseases. After in vivo transplantation, these cells may be exposed to local or systemic inflammation or pathogens, of which they are a common target. Because Toll-like receptors (TLR) are critical in detecting pathogens and in initiating inflammatory responses, we hypothesized that TLR may govern UCB cEPC-derived cells function. While these cells expressed almost all TLR, we found that only TLR3 dramatically impaired cell properties. TLR3 activation inhibited cell proliferation, modified cell cycle entry, impaired the in vitro angiogenic properties and induced pro-inflammatory cytokines production. The anti-angiogenic effect of TLR3 activation was confirmed in vivo in a hind-limb ischemic mice model. Moreover, TLR3 activation consistently leads to an upregulation of miR-29b, -146a and -155 and to a deregulation of cytoskeleton and cell cycle regulator. Hence, TLR3 activation is likely to be a key regulator of cEPC-derived cells properties. PMID:23748743

Grelier, Aurore; Cras, Audrey; Balitrand, Nicole; Delmau, Catherine; Lecourt, Séverine; Lepelletier, Yves; Riesterer, Hélčne; Freida, Delphine; Lataillade, Jean-Jacques; Lebousse-Kerdiles, Marie-Caroline; Cuccini, Wendy; Peffault de Latour, Regis; Marolleau, Jean-Pierre; Uzan, Georges; Larghero, Jérôme; Vanneaux, Valérie

2013-06-08

385

Structure-function studies of STAR family Quaking proteins bound to their in vivo RNA target sites  

PubMed Central

Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins.

Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J.

2013-01-01

386

Effects of sulfamethoxazole and trimethoprim on human neutrophil and lymphocyte functions in vitro: in vivo effects of co-trimoxazole.  

PubMed Central

The effects of sulfamethoxazole and trimethoprim individually and in combination on in vitro neutrophil random migration, chemotaxis to autologous endotoxin-activated serum and the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, phagocytosis and postphagocytic Nitro Blue Tetrazolium reduction, glycolysis, hexose monophosphate shunt activity, myeloperoxidase-mediated protein iodination, hydrogen peroxide production, and degranulation were assessed. The effects on lymphocyte mitogen-induced transformation were also evaluated. It was found that the test agents individually and in combination at high concentrations (> 100 microgram/ml) caused the inhibition of neutrophil postphagocytic myeloperoxidase-mediated protein iodination, which was related to the interference with H2O2 formation as the enzyme per se was unaffected. Both agents caused the inhibition of lymphocyte transformation at high concentrations (> 100 microgram/ml). In vivo studies before and after the ingestion of co-trimoxazole by three individuals showed no inhibition of any of the neutrophil functions tested. The inhibition of lymphocyte transformation was observed in one individual after the ingestion of the chemotherapeutic agent. These findings indicate that the concentrations which inhibit neutrophil H2O2 production and lymphocyte transformation in vitro are not attainable in vivo.

Anderson, R; Grabow, G; Oosthuizen, R; Theron, A; Van Rensburg, A J

1980-01-01

387

In vivo two-photon uncaging of glutamate revealing the structure-function relationships of dendritic spines in the neocortex of adult mice  

PubMed Central

Abstract Two-photon (2P) uncaging of caged neurotransmitters can efficiently stimulate individual synapses and is widely used to characterize synaptic functions in brain slice preparations. Here we extended 2P uncaging to neocortical pyramidal neurons in adult mice in vivo where caged glutamate was applied from the pial surface. To validate the methodology, we applied a small fluorescent probe using the same method, and confirmed that its concentrations were approximately homogenous up to 200 ?m below the cortical surface, and that the extracellular space of the neocortex was as large as 22%. In fact, in vivo whole-cell recording revealed that 2P glutamate uncaging could elicit transient currents (2pEPSCs) very similar to excitatory postsynaptic currents (EPSCs). A spatial resolution of glutamate uncaging was 0.6–0.8 ?m up to the depth of 200 ?m, and in vivo 2P uncaging was able to stimulate single identified spines. Automated three-dimensional (3-D) mapping of such 2pEPSCs which covered the surfaces of dendritic branches revealed that functional AMPA receptor expression was stable and proportional to spine volume. Moreover, in vivo 2P Ca2+ imaging and uncaging suggested that the amplitudes of glutamate-induced Ca2+ transients were inversely proportional to spine volume. Thus, the key structure–function relationships hold in dendritic spines in adult neocortex in vivo, as in young hippocampal slice preparations. In vivo 2P uncaging will be a powerful tool to investigate properties of synapses in the neocortex.

Noguchi, Jun; Nagaoka, Akira; Watanabe, Satoshi; Ellis-Davies, Graham C R; Kitamura, Kazuo; Kano, Masanobu; Matsuzaki, Masanori; Kasai, Haruo

2011-01-01

388

P-31-NMR analysis of in vivo metabolic events in lymphoma during RIT  

SciTech Connect

Radioimmunotherapy (RIT) is potentially a powerful method for treatment of cancer. The efficacy of radioimmunopharmaceuticals on target tissue were measured serially, in vivo, in the human non-Hodgkin's lymphoma (RAJI tumor) using P-31 surface coil NMR. During application of a pulse dosage at curative levels I-131-labeled monoclonal antibody (Lym-1) specific for this tumor, approximately 10,000 rads were delivered to the tumor and 3,000 to the kidney. ATP/Pi ratios and tissue pH were determined in these tumors during therapy and correlated with tumor volumes and radiation-induced tissue damage. During the course of these experiments, the authors noted that the effects of the radiation dose, calculated to be curative, are non-uniform throughout the tissue mass. Therefore, regions of the sample have been distinguished on the basis of differing magnetization precessional rates around the spatially dependent B/sub 1/ field. Two-dimensional FT processing yielded a metabolite map as a set of spectra deployed as a function of mapping distance from the surface coil. In this fashion, metabolites within the tissue slice can be spatially defined. This noninvasive determination of metabolic function appears useful in characterizing the extent of radiation-induced necrosis, spontaneous recurrence of tumor tissue, and sequential evaluation of various dose modalities. These data will be useful in optimization of human RIT regimens.

Adams, D.A.; DeNardo, G.L.; DeNardo, S.J.

1985-05-01

389

Lens Complementation System for the Genetic Analysis of Growth, Differentiation, and Apoptosis in vivo  

Microsoft Academic Search

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that

Nanette J. Liegeois; James W. Horner; Ronald A. Depinho

1996-01-01

390

BASE Flexible Array Preliminary Receiver Function Analysis  

NASA Astrophysics Data System (ADS)

We present high-density, high-resolution receiver function (RF) images of the Bighorn Mountains of north central Wyoming, to gain insight into the subsurface seismic structures of the range, as part of the Bighorn Arch Seismic Experiment (BASE). Our data set contains over 220 three component seismic stations in the Bighorns region, in some areas with spacing less than 5 km. BASE is a Flexible Array experiment integrated with Earthscope. In order to investigate the Bighorns, a large-scale deployment of seismic instrumentation was deployed in the summers of 2009 and 2010. This included 38 broadband and 172 short period seismic stations, as well as both passive and active source ‘Texan’ deployments. Stations were placed to both densify the already present Transportable Array network as well as to create 5 linear transects. Station spacing along these transects range from four to ten kilometers, crossing the Bighorn Basin, through the Bighorn Arch, and into the Powder River Basin. The main objective of the BASE project is to better understand the tectonic processes involved in the formation of basement-cored arches. The formation of these structures remains a key unsolved tectonic problem. The Bighorn Mountains are an archetype of basement-involved foreland arches and therefore act as an excellent setting for the investigation of these types of structures. Four main formation models have been proposed for the Bighorns, each with unique crustal structures. Through a complete structural analysis of the range, relying heavily on seismic subsurface imaging, it will be possible to determine which of these models best fit observations. Moho topography is a crucial component in supporting these hypotheses, and should be well resolved with RF imaging. In this study P-S wave RFs are used to image the structures beneath the Bighorn Mountains. We present ideas for modeling and filtering approaches to dampen low velocity sedimentary layer reverberations in the Powder River and Bighorn basins, which can mask deeper structure (a problem increasingly affecting EarthScope seismic data as deployments move eastwards). Due to the large number of three component stations placed along transects, Common Conversion Point (CCP) stacking of the receiver function gives a high-resolution 2D slice of the crustal structure. Constraints from the active source ‘Texan’ experiment provide independent P and S wave velocities and allow for a more accurate structural depth estimates in CCP images. Our results will be used to generate a Moho map of the Bighorn region which will be input into a 4D (3D geometry + time) model for foreland arch formation.

Yeck, W. L.; Sheehan, A. F.; Schulte-Pelkum, V.; Yang, Z.; Anderson, M. L.; Erslev, E.

2010-12-01

391

A multifunctional turnip crinkle virus replication enhancer revealed by in vivo functional SELEX.  

PubMed

The motif1-hairpin (M1H), located on (-)-strands of Turnip Crinkle Virus (TCV)-associated satellite RNA C (satC), is a replication enhancer and recombination hotspot. Results of in vivo genetic selection (SELEX: systematic evolution of ligands by exponential enrichment), where 28 bases of the M1H were randomized and then subjected to selection in plants, revealed that most winners contained one to three short motifs, many of which in their (-)-sense orientation are found in TCV and satC (-)-strand promoter elements. Ability to replicate in protoplasts correlated with fitness to accumulate in plants with one significant exception. Winner UC, containing only a seven-base replacement sequence, was the second most fit winner, yet replicated no better than a 28-base random replacement sequence. Fitness of satC containing different M1H replacement sequences could be due to enhanced satC replication or enhanced ability to affect TCV movement, since satC interferes with TCV virion accumulation, which is correlated with enhanced movement to younger tissue. Cells inoculated with TCV and UC accumulated fewer virions when compared to other winners that replicated better in protoplasts but were less fit in plants. UC, and other first and second round winners, contained structures that were on average 33% more stable in their (+)-strand orientation, and most formed hairpins with a A-rich sequence at the base. These results suggest that M1H replacement sequences contribute to the fit