Covert spatial attention is functionally intact in amblyopic human adults

PubMed Central

Roberts, Mariel; Cymerman, Rachel; Smith, R. Theodore; Kiorpes, Lynne; Carrasco, Marisa

2016-01-01

Certain abnormalities in behavioral performance and neural signaling have been attributed to a deficit of visual attention in amblyopia, a neurodevelopmental disorder characterized by a diverse array of visual deficits following abnormal binocular childhood experience. Critically, most have inferred attention's role in their task without explicitly manipulating and measuring its effects against a baseline condition. Here, we directly investigate whether human amblyopic adults benefit from covert spatial attention—the selective processing of visual information in the absence of eye movements—to the same degree as neurotypical observers. We manipulated both involuntary (Experiment 1) and voluntary (Experiment 2) attention during an orientation discrimination task for which the effects of covert spatial attention have been well established in neurotypical and special populations. In both experiments, attention significantly improved accuracy and decreased reaction times to a similar extent (a) between the eyes of the amblyopic adults and (b) between the amblyopes and their age- and gender-matched controls. Moreover, deployment of voluntary attention away from the target location significantly impaired task performance (Experiment 2). The magnitudes of the involuntary and voluntary attention benefits did not correlate with amblyopic depth or severity. Both groups of observers showed canonical performance fields (better performance along the horizontal than vertical meridian and at the lower than upper vertical meridian) and similar effects of attention across locations. Despite their characteristic low-level vision impairments, covert spatial attention remains functionally intact in human amblyopic adults. PMID:28033433

Midbrain stimulation-evoked lumbar spinal activity in the adult decerebrate mouse.

PubMed

Stecina, Katinka

2017-08-15

Genetic techniques rendering murine models a popular choice for neuroscience research has led to important insights on neural networks controlling locomotor function. Using genetically altered mouse models for in vivo, electrophysiological studies in the adult state could validate key principles of locomotor network organization that have been described in neonatal, in vitro preparations. The experimental model presented here describes a decerebrate, in vivo adult mouse preparation in which focal, electrical midbrain stimulation was combined with monitoring lumbar neural activity and motor output after pre-collicular decerebration and neuromuscular blockade. Lumbar cord dorsum potentials (in 9/10 animals) and motoneuron output (in 3/5 animals) including fictive locomotion, was achieved by focal midbrain stimulation. The stimulation electrode locations could be reconstructed (in 6/7 animals) thereby allowing anatomical identification of the stimulated supraspinal regions. This preparation allows for concomitant recording or stimulation in the spinal cord and in the mid/hindbrain of adult mice. It differs from other methods used in the past with adult mice as it does not require pharmacological manipulation of neural excitability in order to generate motor output. Midbrain stimulation can consistently be used for inducing lumbar neural activity in adult mice under neuromuscular blockade. This model is suited for examination of brain-spinal connectivity and it may benefit a wide range of fields depending on the features of the genetically modified mouse models used in combination with the presented methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes

PubMed Central

Nickerson, John M.; Goodman, Penny; Chrenek, Micah A.; Johnson, Christiana J.; Berglin, Lennart; Redmond, T. Michael.; Boatright, Jeffrey H.

2013-01-01

Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 microliters in the human eye and less than 1 microliter in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past ten years (1). PMID:22688698

A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

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

Webb, Carol F., E-mail: carol-webb@omrf.org; Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK; Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights:more » • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.« less

The Adult Mouse Anatomical Dictionary: a tool for annotating and integrating data

PubMed Central

Hayamizu, Terry F; Mangan, Mary; Corradi, John P; Kadin, James A; Ringwald, Martin

2005-01-01

We have developed an ontology to provide standardized nomenclature for anatomical terms in the postnatal mouse. The Adult Mouse Anatomical Dictionary is structured as a directed acyclic graph, and is organized hierarchically both spatially and functionally. The ontology will be used to annotate and integrate different types of data pertinent to anatomy, such as gene expression patterns and phenotype information, which will contribute to an integrated description of biological phenomena in the mouse. PMID:15774030

Endogenous spatial attention: evidence for intact functioning in adults with autism

PubMed Central

Grubb, Michael A.; Behrmann, Marlene; Egan, Ryan; Minshew, Nancy J.; Carrasco, Marisa; Heeger, David J.

2012-01-01

Lay Abstract Attention allows us to selectively process the vast amount of information with which we are confronted. Focusing on a certain location of the visual scene (visual spatial attention) enables the prioritization of some aspects of information while ignoring others. Rapid manipulation of the attention field (i.e., the location and spread of visual spatial attention) is a critical aspect of human cognition, and previous research on spatial attention in individuals with autism spectrum disorders (ASD) has produced inconsistent results. In a series of three experiments, we evaluated claims in the literature that individuals with ASD exhibit a deficit in voluntarily controlling the deployment and size of the spatial attention field. We measured how well participants perform a visual discrimination task (accuracy) and how quickly they do so (reaction time), with and without spatial uncertainty (i.e., the lack of predictability concerning the spatial position of the upcoming stimulus). We found that high–functioning adults with autism exhibited slower reactions times overall with spatial uncertainty, but the effects of attention on performance accuracies and reaction times were indistinguishable between individuals with autism and typically developing individuals, in all three experiments. These results provide evidence of intact endogenous spatial attention function in high–functioning adults with ASD, suggesting that atypical endogenous spatial attention cannot be a latent characteristic of autism in general. Scientific Abstract Rapid manipulation of the attention field (i.e., the location and spread of visual spatial attention) is a critical aspect of human cognition, and previous research on spatial attention in individuals with autism spectrum disorders (ASD) has produced inconsistent results. In a series of three psychophysical experiments, we evaluated claims in the literature that individuals with ASD exhibit a deficit in voluntarily controlling the

Resolving stem and progenitor cells in the adult mouse incisor through gene co-expression analysis

PubMed Central

Seidel, Kerstin; Marangoni, Pauline; Tang, Cynthia; Houshmand, Bahar; Du, Wen; Maas, Richard L; Murray, Steven; Oldham, Michael C; Klein, Ophir D

2017-01-01

Investigations into stem cell-fueled renewal of an organ benefit from an inventory of cell type-specific markers and a deep understanding of the cellular diversity within stem cell niches. Using the adult mouse incisor as a model for a continuously renewing organ, we performed an unbiased analysis of gene co-expression relationships to identify modules of co-expressed genes that represent differentiated cells, transit-amplifying cells, and residents of stem cell niches. Through in vivo lineage tracing, we demonstrated the power of this approach by showing that co-expression module members Lrig1 and Igfbp5 define populations of incisor epithelial and mesenchymal stem cells. We further discovered that two adjacent mesenchymal tissues, the periodontium and dental pulp, are maintained by distinct pools of stem cells. These findings reveal novel mechanisms of incisor renewal and illustrate how gene co-expression analysis of intact biological systems can provide insights into the transcriptional basis of cellular identity. DOI: http://dx.doi.org/10.7554/eLife.24712.001 PMID:28475038

Skin test sensitivity to mouse predicts allergic symptoms to nasal challenge in urban adults.

PubMed

Chong, Laura K; Ong, Mary Jane; Curtin-Brosnan, Jean; Matsui, Elizabeth C

2010-01-01

Epidemiologic studies have shown an association between mouse allergen exposure and asthma morbidity among urban populations, but confirmatory challenge studies in community populations have not been performed. This study was designed to examine the clinical relevance of mouse sensitization using a nasal challenge model. Forty-nine urban adults with asthma underwent skin-prick testing (SPT) and intradermal testing (IDT) with mouse epithelia extract. A positive SPT was defined as a net wheal size ≥3 mm and a positive IDT was defined as a net wheal size ≥6 mm using a 1:100 dilution of extract (1:10 w/v was obtained from Greer Laboratories (Lenoir, NC) as a single lot [Mus m 1 concentration = 2130 ng/mL]). Mouse-specific IgE (m-IgE) was measured by ImmunoCAP (Phadia, Uppsala, Sweden). Nasal challenge was performed with increasing concentrations of mouse epithelia extract and symptoms were assessed by visual analog scale. A positive challenge was defined as a 20-mm increase in the scale. The age range of the 49 participants was 18-50 years; 41% were men and 86% were black. Fourteen participants were SPT(+) to mouse, 15 participants were SPT(-) but (IDT(+)), and 20 participants were negative on both SPT(-) and IDT(-) (SPT(-)/IDT(-)). Sixty-four percent of the SPT(+) group, 40% of the IDT(+) group, and 20% of the SPT(-)/IDT(-) group had a positive nasal challenge. Sixty-seven percent (10/15) of those who were either SPT(+) or m-IgE(+) had a positive nasal challenge. SPT or the combination of SPT plus m-IgE performed best in diagnosing mouse allergy. The great majority of mouse-sensitized urban adults with asthma appear to have clinically relevant sensitization. Urban adults with asthma should be evaluated for mouse sensitization using SPT or SPT plus m-IgE testing.

Prolactin Stimulates Precursor Cells in the Adult Mouse Hippocampus

PubMed Central

Walker, Tara L.; Vukovic, Jana; Koudijs, Margaretha M.; Blackmore, Daniel G.; Mackay, Eirinn W.; Sykes, Alex M.; Overall, Rupert W.; Hamlin, Adam S.; Bartlett, Perry F.

2012-01-01

In the search for ways to combat degenerative neurological disorders, neurogenesis-stimulating factors are proving to be a promising area of research. In this study, we show that the hormonal factor prolactin (PRL) can activate a pool of latent precursor cells in the adult mouse hippocampus. Using an in vitro neurosphere assay, we found that the addition of exogenous PRL to primary adult hippocampal cells resulted in an approximate 50% increase in neurosphere number. In addition, direct infusion of PRL into the adult dentate gyrus also resulted in a significant increase in neurosphere number. Together these data indicate that exogenous PRL can increase hippocampal precursor numbers both in vitro and in vivo. Conversely, PRL null mice showed a significant reduction (approximately 80%) in the number of hippocampal-derived neurospheres. Interestingly, no deficit in precursor proliferation was observed in vivo, indicating that in this situation other niche factors can compensate for a loss in PRL. The PRL loss resulted in learning and memory deficits in the PRL null mice, as indicated by significant deficits in the standard behavioral tests requiring input from the hippocampus. This behavioral deficit was rescued by direct infusion of recombinant PRL into the hippocampus, indicating that a lack of PRL in the adult mouse hippocampus can be correlated with impaired learning and memory. PMID:22973440

The Changing Sensory and Sympathetic Innervation of the Young, Adult and Aging Mouse Femur.

PubMed

Chartier, Stephane R; Mitchell, Stefanie A T; Majuta, Lisa A; Mantyh, Patrick W

2018-02-10

Although bone is continually being remodeled and ultimately declines with aging, little is known whether similar changes occur in the sensory and sympathetic nerve fibers that innervate bone. Here, immunohistochemistry and confocal microscopy were used to examine changes in the sensory and sympathetic nerve fibers that innervate the young (10 days post-partum), adult (3 months) and aging (24 months) C57Bl/6 mouse femur. In all three ages examined, the periosteum was the most densely innervated bone compartment. With aging, the total number of sensory and sympathetic nerve fibers clearly declines as the cambium layer of the periosteum dramatically thins. Yet even in the aging femur, there remains a dense sensory and sympathetic innervation of the periosteum. In cortical bone, sensory and sympathetic nerve fibers are largely confined to vascularized Haversian canals and while there is no significant decline in the density of sensory fibers, there was a 75% reduction in sympathetic nerve fibers in the aging vs. adult cortical bone. In contrast, in the bone marrow the overall density/unit area of both sensory and sympathetic nerve fibers appeared to remain largely unchanged across the lifespan. The preferential preservation of sensory nerve fibers suggests that even as bone itself undergoes a marked decline with age, the nociceptors that detect injury and signal skeletal pain remain relatively intact. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Histology and ultrastructure of transitional changes in skin morphology in the juvenile and adult four-striped mouse (Rhabdomys pumilio).

PubMed

Stewart, Eranée; Ajao, Moyosore Salihu; Ihunwo, Amadi Ogonda

2013-01-01

The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin.

Dissection of complex adult traits in a mouse synthetic population.

PubMed

Burke, David T; Kozloff, Kenneth M; Chen, Shu; West, Joshua L; Wilkowski, Jodi M; Goldstein, Steven A; Miller, Richard A; Galecki, Andrzej T

2012-08-01

Finding the causative genetic variations that underlie complex adult traits is a significant experimental challenge. The unbiased search strategy of genome-wide association (GWAS) has been used extensively in recent human population studies. These efforts, however, typically find only a minor fraction of the genetic loci that are predicted to affect variation. As an experimental model for the analysis of adult polygenic traits, we measured a mouse population for multiple phenotypes and conducted a genome-wide search for effector loci. Complex adult phenotypes, related to body size and bone structure, were measured as component phenotypes, and each subphenotype was associated with a genomic spectrum of candidate effector loci. The strategy successfully detected several loci for the phenotypes, at genome-wide significance, using a single, modest-sized population (N = 505). The effector loci each explain 2%-10% of the measured trait variation and, taken together, the loci can account for over 25% of a trait's total population variation. A replicate population (N = 378) was used to confirm initially observed loci for one trait (femur length), and, when the two groups were merged, the combined population demonstrated increased power to detect loci. In contrast to human population studies, our mouse genome-wide searches find loci that individually explain a larger fraction of the observed variation. Also, the additive effects of our detected mouse loci more closely match the predicted genetic component of variation. The genetic loci discovered are logical candidates for components of the genetic networks having evolutionary conservation with human biology.

Sox2 and Jagged1 Expression in Normal and Drug-Damaged Adult Mouse Inner Ear

PubMed Central

Campbell, Sean; Taylor, Ruth R.; Forge, Andrew; Hume, Clifford R.

2007-01-01

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear. PMID:18157569

Regulation by commensal bacteria of neurogenesis in the subventricular zone of adult mouse brain.

PubMed

Sawada, Naoki; Kotani, Takenori; Konno, Tasuku; Setiawan, Jajar; Nishigaito, Yuka; Saito, Yasuyuki; Murata, Yoji; Nibu, Ken-Ichi; Matozaki, Takashi

2018-04-15

In the mouse olfactory bulb (OB), interneurons such as granule cells and periglomerular cells are continuously replaced by adult-born neurons, which are generated in the subventricular zone (SVZ) of the brain. We have now investigated the role of commensal bacteria in regulation of such neuronal cell turnover in the adult mouse brain. Administration of mixture of antibiotics to specific pathogen-free (SPF) mice markedly attenuated the incorporation of bromodeoxyuridine (BrdU) into the SVZ cells. The treatment with antibiotics also reduced newly generated BrdU-positive neurons in the mouse OB. In addition, the incorporation of BrdU into the SVZ cells of germ-free (GF) mice was markedly reduced compared to that apparent for SPF mice. In contrast, the reduced incorporation of BrdU into the SVZ cells of GF mice was recovered by their co-housing with SPF mice, suggesting that commensal bacteria promote the incorporation of BrdU into the SVZ cells. Finally, we found that administration of ampicillin markedly attenuated the incorporation of BrdU into the SVZ cells of SPF mice. Our results thus suggest that ampicillin-sensitive commensal bacteria regulate the neurogenesis in the SVZ of adult mouse brain. Copyright © 2018 Elsevier Inc. All rights reserved.

Colocalization properties of elementary Ca(2+) release signals with structures specific to the contractile filaments and the tubular system of intact mouse skeletal muscle fibers.

PubMed

Georgiev, Tihomir; Zapiec, Bolek; Förderer, Moritz; Fink, Rainer H A; Vogel, Martin

2015-12-01

Ca(2+) regulates several important intracellular processes. We combined second harmonic generation (SHG) and two photon excited fluorescence microscopy (2PFM) to simultaneously record the SHG signal of the myosin filaments and localized elementary Ca(2+) release signals (LCSs). We found LCSs associated with Y-shaped structures of the myosin filament pattern (YMs), so called verniers, in intact mouse skeletal muscle fibers under hypertonic treatment. Ion channels crucial for the Ca(2+) regulation are located in the tubular system, a system that is important for Ca(2+) regulation and excitation-contraction coupling. We investigated the tubular system of intact, living mouse skeletal muscle fibers using 2PFM and the fluorescent Ca(2+) indicator Fluo-4 dissolved in the external solution or the membrane dye di-8-ANEPPS. We simultaneously measured the SHG signal from the myosin filaments of the skeletal muscle fibers. We found that at least a subset of the YMs observed in SHG images are closely juxtaposed with Y-shaped structures of the transverse tubules (YTs). The distances of corresponding YMs and YTs yield values between 1.3 μm and 4.1 μm including pixel uncertainty with a mean distance of 2.52±0.10 μm (S.E.M., n=41). Additionally, we observed that some of the linear-shaped areas in the tubular system are colocalized with linear-shaped areas in the SHG images. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of long-term label-retaining cells in the regeneration of adult mouse kidney after ischemia/reperfusion injury.

PubMed

Liu, Xiangchun; Liu, Haiying; Sun, Lina; Chen, Zhixin; Nie, Huibin; Sun, Aili; Liu, Gang; Guan, Guangju

2016-04-30

Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration. We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU(+)LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU(+)LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU(+)LRCs in mice after ischemia/reperfusion (I/R) injury. The findings revealed that the nuclei of BrdU(+) LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU(+) LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU(+) LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney. Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered.

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies.

PubMed

Feigenspan, Andreas; Babai, Norbert Zsolt

2017-01-27

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue. To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows (1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites. Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

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

Patel, Shreya, E-mail: Shreya.patel214@gmail.com; Peretz, Jackye, E-mail: Jackye.peretz@gmail.com; Pan, Yuan-Xiang, E-mail: yxpan@illinois.edu

Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 micemore » were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes. �

A double-blind, placebo-controlled, randomized trial of Ginkgo biloba extract EGb 761 in a sample of cognitively intact older adults: neuropsychological findings.

PubMed

Mix, Joseph A; Crews, W David

2002-08-01

There appears to be an absence of large-scaled clinical trials that have examined the efficacy of Ginkgo biloba extract on the neuropsychological functioning of cognitively intact older adults. The importance of such clinical research appears paramount in light of the plethora of products containing Ginkgo biloba that are currently being widely marketed to predominantly cognitively intact adults with claims of enhanced cognitive performances. The purpose of this research was to conduct the first known, large-scaled clinical trial of the efficacy of Ginkgo biloba extract (EGb 761) on the neuropsychological functioning of cognitively intact older adults. Two hundred and sixty-two community-dwelling volunteers (both male and female) 60 years of age and older, who reported no history of dementia or significant neurocognitive impairments and obtained Mini-Mental State Examination total scores of at least 26, were examined via a 6-week, randomized, double-blind, fixed-dose, placebo-controlled, parallel-group, clinical trial. Participants were randomly assigned to receive either Ginkgo biloba extract EGb 761(n = 131; 180 mg/day) or placebo (n = 131) for 6 weeks. Efficacy measures consisted of participants' raw change in performance scores from pretreatment baseline to those obtained just prior to termination of treatment on the following standardized neuropsychological measures: Selective Reminding Test (SRT), Wechsler Adult Intelligence Scale-III Block Design (WAIS-III BD) and Digit Symbol-Coding (WAIS-III DS) subtests, and the Wechsler Memory Scale-III Faces I (WMS-III FI) and Faces II (WMS-III FII) subtests. A subjective Follow-up Self-report Questionnaire was also administered to participants just prior to termination of the treatment phase. Analyses of covariance indicated that cognitively intact participants who received 180 mg of EGb 761 daily for 6 weeks exhibited significantly more improvement on SRT tasks involving delayed (30 min) free recall (p < 0.04) and

Fluoxetine increases plasticity and modulates the proteomic profile in the adult mouse visual cortex

PubMed Central

Ruiz-Perera, L.; Muniz, M.; Vierci, G.; Bornia, N.; Baroncelli, L.; Sale, A.; Rossi, F.M.

2015-01-01

The scarce functional recovery of the adult CNS following injuries or diseases is largely due to its reduced potential for plasticity, the ability to reorganize neural connections as a function of experience. Recently, some new strategies restoring high levels of plasticity in the adult brain have been identified, especially in the paradigmatic model of the visual system. A chronic treatment with the anti-depressant fluoxetine reinstates plasticity in the adult rat primary visual cortex, inducing recovery of vision in amblyopic animals. The molecular mechanisms underlying this effect remain largely unknown. Here, we explored fluoxetine effects on mouse visual cortical plasticity, and exploited a proteomic approach to identify possible candidates mediating the outcome of the antidepressant treatment on adult cortical plasticity. We showed that fluoxetine restores ocular dominance plasticity in the adult mouse visual cortex, and identified 31 differentially expressed protein spots in fluoxetine-treated animals vs. controls. MALDITOF/TOF mass spectrometry identification followed by bioinformatics analysis revealed that these proteins are involved in the control of cytoskeleton organization, endocytosis, molecular transport, intracellular signaling, redox cellular state, metabolism and protein degradation. Altogether, these results indicate a complex effect of fluoxetine on neuronal signaling mechanisms potentially involved in restoring plasticity in the adult brain. PMID:26205348

Narratives of Self-Neglect: Patterns of Traumatic Personal Experiences and Maladaptive Behaviors in Cognitively Intact Older Adults.

PubMed

Lien, Cynthia; Rosen, Tony; Bloemen, Elizabeth M; Abrams, Robert C; Pavlou, Maria; Lachs, Mark S

2016-11-01

To identify patterns of personal experience or behavior in self-neglect by exploring narratives of cognitively intact older adults. Descriptive study involving semistructured interviews and unstructured narratives. A parent study of self-neglect characteristics. Cognitively intact, self-neglecting older adults referred from 11 community-based senior services agencies (N = 69). Interviews included a comprehensive psychiatric assessment using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Axis-I and II Disorders and an unstructured interview that allowed subjects to describe important elements of their life stories. Content analysis was used to identify personal experiences and behavior patterns in each subject's narrative. Four types of traumatic personal experiences (psychologically traumatic loss, separation or abandonment (29%); violent victimization, physical trauma, or sexual abuse (19%); exposure to war or political violence (9%); prolonged mourning (7%)) and five behavior patterns (significant financial instability (23%), severe lifelong mental illness (16%), mistrust of people or paranoia (13%), distrust and avoidance of the medical establishment (13%), substance abuse or dependence (13%)) were identified in the life stories. Patterns of traumatic personal experiences and maladaptive behaviors that self-neglecters frequently report were identified. Experiences, perceptions, and behaviors developed over a lifetime may contribute to elder self-neglect. Further exploration and better understanding of these patterns may identify potential risk factors and areas for future targeted screening, intervention, and prevention. © 2016, Copyright the Authors Journal compilation © 2016, The American Geriatrics Society.

Uptake of ingested bovine lactoferrin and its accumulation in adult mouse tissues.

PubMed

Fischer, Romy; Debbabi, Hajer; Blais, Anne; Dubarry, Michel; Rautureau, Michèle; Boyaka, Prosper N; Tome, Daniel

2007-10-01

Lactoferrin is a glycoprotein with antimicrobial and immunoregulatory properties, which is found in milk, other external secretions, and in the secondary granules of neutrophils. The present study examined the time course of uptake and the pattern of tissue accumulation of bovine lactoferrin (bLf) following intragastric intubation of a single dose to adult naïve mice or to mice daily fed bLf for 4 weeks. Following ingestion, bLf was transferred from the intestine into peripheral blood in a form with intact molecular weight (80 kDa) and localized within 10 to 20 min after oral administration in the liver, kidneys, gall bladder, spleen, and brain of both groups of mice. Immunoreactive bLf could also be detected in the luminal contents of the stomach, small intestine and colon 1 h after intragastric intubation. Interestingly, serum and tissue accumulation of bLf was approximately 50% lower in mice chronically fed this protein than in those given only the single oral dose. Furthermore, significant levels of bLf-specific IgA and IgG antibodies as well as bLf-containing IgA- and IgG immune complexes were detected in mice chronically fed bLf but not in those fed only once. Taken together, these results indicate that bLf resists major proteolytic degradation in the intestinal lumen and is readily absorbed in an antigenic form in blood and various mouse tissues. Chronic ingestion of lactoferrin reduces its uptake, probably through mechanisms such as immune exclusion, which minimize potential harmful reactions to food products.

The RNA-binding protein Musashi-1 is produced in the developing and adult mouse eye.

PubMed

Raji, B; Dansault, A; Leemput, J; de la Houssaye, G; Vieira, V; Kobetz, A; Arbogast, L; Masson, C; Menasche, M; Abitbol, M

2007-08-10

Musashi-1 (Msi1) is an RNA-binding protein produced in various types of stem cells including neural stem/progenitor cells and astroglial progenitor cells in the vertebrate central nervous system. Other RNA-binding proteins such as Pumilio-1, Pumilio-2, Staufen-1, and Staufen-2 have been characterized as potential markers of several types of stem or progenitor cells. We investigated the involvement of Msi1 in mouse eye development and adult mouse eye functions by analyzing the profile of Msi1 production in all ocular structures during development and adulthood. We studied Msi1 production by in situ hybridization and immunohistochemistry of ocular tissue sections and by semi-quantitative RT-PCR and western blot analysis from the embryonic stage of 12.5 days post coitum (E12.5 dpc) when the first retinal ganglion cells (RGCs) begin to appear to the adult stage when all retinal cell types are present. Msi1 mRNA was present at all studied stages of eye development. Msi1 protein was detected in the primitive neuroblastic layer (NbL), the ganglion cell layer (GCL), and in all major differentiated neurons of postnatal developing and adult retinae. During postnatal developing stages, faint diffuse Msi1 protein staining is converted to a more specific distribution once mouse retina is fully differentiated. The most striking result of our study concerns the large amounts of Msi1 protein and mRNA in several unexpected sites of adult mouse eyes including the corneal epithelium and endothelium, stromal keratocytes, progenitor cells of the limbus, equatorial lens stem cells, differentiated lens epithelial cells, and differentiating lens fibers. Msi1 was also found in the pigmented and nonpigmented cells of the ciliary processes, the melanocytes of the ciliary body, the retinal pigment epithelium, differentiated retinal neurons, and most probably in the retinal glial cells such as Müller glial cells, astrocytes, and the oligodendocytes surrounding the axons of the optic nerve

Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.

PubMed

Hou, Sheng T; Dove, Mike; Anderson, Erica; Zhang, Jiangbing; MacKenzie, C Roger

2004-09-30

Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.

Adult mouse brain gene expression patterns bear an embryologic imprint

PubMed Central

Zapala, Matthew A.; Hovatta, Iiris; Ellison, Julie A.; Wodicka, Lisa; Del Rio, Jo A.; Tennant, Richard; Tynan, Wendy; Broide, Ron S.; Helton, Rob; Stoveken, Barbara S.; Winrow, Christopher; Lockhart, Daniel J.; Reilly, John F.; Young, Warren G.; Bloom, Floyd E.; Lockhart, David J.; Barlow, Carrolee

2005-01-01

The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional “imprint” consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior–posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org). PMID:16002470

Malnutrition among Cognitively Intact, non-Critically Ill Older Adults in the Emergency Department

PubMed Central

Pereira, Greg F.; Bulik, Cynthia M.; Weaver, Mark A.; Holland, Wesley C.; Platts-Mills, Timothy F.

2014-01-01

Objectives We estimate the prevalence of malnutrition among older patients presenting to an emergency department (ED) in the southeastern United States and identify subgroups at increased risk. Methods We conducted a cross-sectional study with random time block sampling of cognitively intact patients aged 65 years and older. Nutrition was assessed using the Mini Nutritional Assessment Short-Form (0–14 scale) with malnutrition defined as a score of 7 or less and at-risk for malnutrition defined as a score of 8–11. The presence of depressive symptoms was defined as a Center for Epidemiological Studies Depression-10 score of 4 or more (0–10 scale). Results Among 138 older adults, 16% (95% Confidence Interval [CI], 10%–22%) were malnourished and 60% (95% CI, 52%–69%) were either malnourished or at-risk for malnutrition. Seventeen of the 22 malnourished patients (77%) denied previously being diagnosed with malnutrition. The prevalence of malnutrition was not appreciably different between males and females, across levels of patient education, or between those living in urban and rural areas. However, the prevalence of malnutrition was higher among patients with depressive symptoms 52%, those residing in assisted living 50%, those with difficulty eating 38%, and those reporting difficulty buying groceries 33%. Conclusion Among a random sample of cognitively intact older ED patients, more than half were malnourished or at-risk for malnutrition, and the majority of malnourished patients had not previously been diagnosed. Higher rates of malnutrition among those with depression, difficulty eating, and difficulty buying groceries suggest the need to explore multifaceted interventions. PMID:25129819

Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse.

PubMed

Mousseau, D D; Larson, A A

1994-09-01

We have previously observed similarities in the behavioral effects produced by the NH2-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl)-guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro2, D-Phe7]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [3H]-DTG binding sites in the mouse brain and spinal cord in vitro: Brain; DTG = HAL > SP = MOR = NAL > SP(1-7) > D-SP(1-7) > SP(5-11): Spinal cord; DTG = HAL > SP(1-7) = MOR = NAL > SP > D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [3H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1-7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site.

An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

PubMed

Schulze, Katja; Galichet, Arnaud; Sayar, Beyza S; Scothern, Anthea; Howald, Denise; Zymann, Hillard; Siffert, Myriam; Zenhäusern, Denise; Bolli, Reinhard; Koch, Peter J; Garrod, David; Suter, Maja M; Müller, Eliane J

2012-02-01

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

PubMed Central

Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

PubMed

Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain

PubMed Central

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889

Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain.

PubMed

Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

2013-01-01

An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain.

Evaluating methods of mouse euthanasia on the oocyte quality: cervical dislocation versus isoflurane inhalation.

PubMed

Roustan, Audrey; Perrin, Jeanne; Berthelot-Ricou, Anaïs; Lopez, Erica; Botta, Alain; Courbiere, Blandine

2012-04-01

Cervical dislocation is a commonly used method of mouse euthanasia. Euthanasia by isoflurane inhalation is an alternative method which allows the sacrifice of several mice at the same time with an anaesthesia, in the aim to decrease pain and animal distress. The objective of our study was to assess the impact of these two methods of euthanasia on the quality of mouse oocytes. By administering gonadotropins, we induced a superovulation in CD1 female mice. Mice were randomly assigned to euthanasia with cervical dislocation and isoflurane inhalation. Oviducts were collected and excised to retrieve metaphase II oocytes. After microscopic examination, oocytes were classified into three groups: intact, fragmented/cleaved and atretic. Intact metaphase II oocytes were employed for biomedical research. A total of 1442 oocytes in the cervical dislocation group were compared with 1230 oocytes in the isoflurane group. In the cervical dislocation group, 93.1% of the oocytes were intact, versus 65.8% in the isoflurane group (P ≤ 0.001). In light of these results, we conclude that cervical dislocation is the best method of mouse euthanasia for obtaining intact oocytes for biomedical research.

A robust method for RNA extraction and purification from a single adult mouse tendon.

PubMed

Grinstein, Mor; Dingwall, Heather L; Shah, Rishita R; Capellini, Terence D; Galloway, Jenna L

2018-01-01

Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of Scx gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze Sox9 and Col1a2 gene expression changes in injured compared with uninjured control tendons. Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

PubMed

Eckardt, D; Theis, M; Degen, J; Ott, T; van Rijen, H V M; Kirchhoff, S; Kim, J-S; de Bakker, J M T; Willecke, K

2004-01-01

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

PubMed

Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

2016-07-29

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

Hypophysectomy eliminates and growth hormone (GH) maintains the midpregnancy elevation in GH receptor and serum binding protein in the mouse

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

Sanchez-Jimenez, F.; Fielder, P.J.; Martinez, R.R.

1990-02-01

({sup 125}I)Iodomouse GH (({sup 125}I)iodo-mGH) binding to samples of serum and hepatic microsomal membranes was measured in hypophysectomized pregnant, sham-operated pregnant, intact pregnant, and intact adult virgin mice. Surgeries were carried out on day 11 of pregnancy, and the animals were killed on day 14. The binding of mGH to both serum and hepatic microsomal membranes of intact virgin mice was much lower than to those of intact pregnant mice. In hypophysectomized mice, the mGH-binding capacity of both serum and hepatic microsomes decreased to values similar to those of nonpregnant mice. No significant differences were observed between intact and sham-operatedmore » pregnant animals in the maternal serum mGH concentration, the serum GH-binding protein concentration, or the hepatic GH receptor concentration. GH receptor and binding protein-encoding mRNAs were also higher in intact and sham-operated pregnant mice than in virgin and hypophysectomized mice. Hypophysectomized mice were treated with 200 micrograms/day bovine GH, administered by osmotic minipump; after 3 days of treatment, a significant elevation of hepatic GH receptor and serum GH-binding protein levels was observed. These results demonstrate an up-regulation of hepatic GH receptors and serum GH-binding protein by GH during pregnancy in the mouse.« less

Localization of cholinergic innervation and neurturin receptors in adult mouse heart and expression of the neurturin gene.

PubMed

Mabe, Abigail M; Hoard, Jennifer L; Duffourc, Michelle M; Hoover, Donald B

2006-10-01

Neurturin (NRTN) is a neurotrophic factor required during development for normal cholinergic innervation of the heart, but whether NRTN continues to function in the adult heart is unknown. We have therefore evaluated NRTN expression in adult mouse heart and the association of NRTN receptors with intracardiac cholinergic neurons and nerve fibers. Mapping the regional distribution and density of cholinergic nerves in mouse heart was an integral part of this goal. Analysis of RNA from adult C57BL/6 mouse hearts demonstrated NRTN expression in atrial and ventricular tissue. Virtually all neurons in the cardiac parasympathetic ganglia exhibited the cholinergic phenotype, and over 90% of these cells contained both components of the NRTN receptor, Ret tyrosine kinase and GDNF family receptor alpha2 (GFRalpha2). Cholinergic nerve fibers, identified by labeling for the high affinity choline transporter, were abundant in the sinus and atrioventricular nodes, ventricular conducting system, interatrial septum, and much of the right atrium, but less abundant in the left atrium. The right ventricular myocardium contained a low density of cholinergic nerves, which were sparse in other regions of the working ventricular myocardium. Some cholinergic nerves were also associated with coronary vessels. GFRalpha2 was present in most cholinergic nerve fibers and in Schwann cells and their processes throughout the heart. Some cholinergic nerve fibers, such as those in the sinus node, also exhibited Ret immunoreactivity. These findings provide the first detailed mapping of cholinergic nerves in mouse heart and suggest that the neurotrophic influence of NRTN on cardiac cholinergic innervation continues in mature animals.

Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

PubMed Central

Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

2016-01-01

In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

PubMed

Guerrero-Bosagna, Carlos; Covert, Trevor R; Haque, Md M; Settles, Matthew; Nilsson, Eric E; Anway, Matthew D; Skinner, Michael K

2012-12-01

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease. Copyright © 2012 Elsevier Inc. All rights reserved.

Epigenetic Transgenerational Inheritance of Vinclozolin Induced Mouse Adult Onset Disease and Associated Sperm Epigenome Biomarkers

PubMed Central

Guerrero-Bosagna, Carlos; Covert, Trevor R.; Haque, Md. M.; Settles, Matthew; Nilsson, Eric E.; Anway, Matthew D.; Skinner, Michael K.

2012-01-01

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease. PMID:23041264

A Simplified, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse Heart

PubMed Central

Ackers-Johnson, Matthew; Li, Peter Yiqing; Holmes, Andrew P.; O’Brien, Sian-Marie; Pavlovic, Davor; Foo, Roger S.

2018-01-01

Rationale Cardiovascular disease represents a global pandemic. The advent of and recent advances in mouse genomics, epigenomics, and transgenics offer ever-greater potential for powerful avenues of research. However, progress is often constrained by unique complexities associated with the isolation of viable myocytes from the adult mouse heart. Current protocols rely on retrograde aortic perfusion using specialized Langendorff apparatus, which poses considerable logistical and technical barriers to researchers and demands extensive training investment. Objective To identify and optimize a convenient, alternative approach, allowing the robust isolation and culture of adult mouse cardiac myocytes using only common surgical and laboratory equipment. Methods and Results Cardiac myocytes were isolated with yields comparable to those in published Langendorff-based methods, using direct needle perfusion of the LV ex vivo and without requirement for heparin injection. Isolated myocytes can be cultured antibiotic free, with retained organized contractile and mitochondrial morphology, transcriptional signatures, calcium handling, responses to hypoxia, neurohormonal stimulation, and electric pacing, and are amenable to patch clamp and adenoviral gene transfer techniques. Furthermore, the methodology permits concurrent isolation, separation, and coculture of myocyte and nonmyocyte cardiac populations. Conclusions We present a novel, simplified method, demonstrating concomitant isolation of viable cardiac myocytes and nonmyocytes from the same adult mouse heart. We anticipate that this new approach will expand and accelerate innovative research in the field of cardiac biology. PMID:27502479

Not GABA but glycine mediates segmental, propriospinal, and bulbospinal postsynaptic inhibition in adult mouse spinal forelimb motor neurons.

PubMed

Jiang, Juan; Alstermark, Bror

2015-02-04

The general view is that both glycine (Eccles, 1964) and GABA (Curtis and Felix, 1971) evoke postsynaptic inhibition in spinal motor neurons. In newborn or juvenile animals, there are conflicting results showing postsynaptic inhibition in motor neurons by corelease of GABA and glycine (Jonas et al., 1998) or by glycine alone (Bhumbra et al., 2012). To resolve the relative contributions of GABA and glycine to postsynaptic inhibition, we performed in vivo intracellular recordings from forelimb motor neurons in adult mice. Postsynaptic potentials evoked from segmental, propriospinal, and bulbospinal systems in motor neurons were compared across four different conditions: control, after gabazine, gabazine followed by strychnine, and strychnine alone. No significant differences were observed in the proportion of IPSPs and EPSPs between control and gabazine conditions. In contrast, EPSPs but not IPSPs were recorded after adding strychnine with gabazine or administering strychnine alone, suggesting an exclusive role for glycine in postsynaptic inhibition. To test whether the injected (intraperitoneal) dose of gabazine blocked GABAergic inhibitory transmission, we evoked GABAA receptor-mediated monosynaptic IPSPs in deep cerebellar nuclei neurons by stimulation of Purkinje cell fibers. No monosynaptic IPSPs could be recorded in the presence of gabazine, showing the efficacy of gabazine treatment. Our results demonstrate that, in the intact adult mouse, the postsynaptic inhibitory effects in spinal motor neurons exerted by three different systems, intrasegmental and intersegmental as well as supraspinal, are exclusively glycinergic. These findings emphasize the importance of glycinergic postsynaptic inhibition in motor neurons and challenge the view that GABA also contributes. Copyright © 2015 the authors 0270-6474/15/351991-08$15.00/0.

Mouse xenograft modeling of human adult acute lymphoblastic leukemia provides mechanistic insights into adult LIC biology

PubMed Central

Dey, Aditi; Castleton, Anna Z.; Schwab, Claire; Samuel, Edward; Sivakumaran, Janani; Beaton, Brendan; Zareian, Nahid; Zhang, Christie Yu; Rai, Lena; Enver, Tariq; Moorman, Anthony V.; Fielding, Adele K.

2014-01-01

The distinct nature of acute lymphoblastic leukemia (ALL) in adults, evidenced by inferior treatment outcome and different genetic landscape, mandates specific studies of disease-initiating mechanisms. In this study, we used NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches to elucidate leukemia-initiating cell (LIC) biology in primary adult precursor B (pre-B) ALL to optimize disease modeling. In contrast with xenografting studies of pediatric ALL, we found that modification of the NSG host environment using preconditioning total body irradiation (TBI) was indispensable for efficient engraftment of adult non-t(4;11) pre-B ALL, whereas t(4;11) pre-B ALL was successfully reconstituted without this adaptation. Furthermore, TBI-based xenotransplantation of non-t(4;11) pre-B ALL enabled detection of a high frequency of LICs (<1:6900) and permitted frank leukemic engraftment from a remission sample containing drug-resistant minimal residual disease. Investigation of TBI-sensitive stromal-derived factor-1/chemokine receptor type 4 signaling revealed greater functional dependence of non-t(4;11) pre-B ALL on this niche-based interaction, providing a possible basis for the differential engraftment behavior. Thus, our studies establish the optimal conditions for experimental modeling of human adult pre-B ALL and demonstrate the critical protumorogenic role of microenvironment-derived SDF-1 in regulating adult pre-B LIC activity that may present a therapeutic opportunity. PMID:24825861

Arc restores juvenile plasticity in adult mouse visual cortex

PubMed Central

Jenks, Kyle R.; Kim, Taekeun; Pastuzyn, Elissa D.; Okuno, Hiroyuki; Taibi, Andrew V.; Bear, Mark F.

2017-01-01

The molecular basis for the decline in experience-dependent neural plasticity over age remains poorly understood. In visual cortex, the robust plasticity induced in juvenile mice by brief monocular deprivation during the critical period is abrogated by genetic deletion of Arc, an activity-dependent regulator of excitatory synaptic modification. Here, we report that augmenting Arc expression in adult mice prolongs juvenile-like plasticity in visual cortex, as assessed by recordings of ocular dominance (OD) plasticity in vivo. A distinguishing characteristic of juvenile OD plasticity is the weakening of deprived-eye responses, believed to be accounted for by the mechanisms of homosynaptic long-term depression (LTD). Accordingly, we also found increased LTD in visual cortex of adult mice with augmented Arc expression and impaired LTD in visual cortex of juvenile mice that lack Arc or have been treated in vivo with a protein synthesis inhibitor. Further, we found that although activity-dependent expression of Arc mRNA does not change with age, expression of Arc protein is maximal during the critical period and declines in adulthood. Finally, we show that acute augmentation of Arc expression in wild-type adult mouse visual cortex is sufficient to restore juvenile-like plasticity. Together, our findings suggest a unifying molecular explanation for the age- and activity-dependent modulation of synaptic sensitivity to deprivation. PMID:28790183

A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

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

Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

1994-09-01

The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing tomore » the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and

Functional plasticity of regenerated and intact taste receptors in adult rats unmasked by dietary sodium restriction.

PubMed

Hill, D L; Phillips, L M

1994-05-01

Unilateral chorda tympani nerve sectioning was combined with institution of a sodium-restricted diet in adult rats to determine the role that environment has on the functional properties of regenerating taste receptor cells. Rats receiving chorda tympani sectioning but no dietary manipulation (cut controls) and rats receiving only the dietary manipulation (diet controls) had normal responses to a concentration series of NaCl, sodium acetate (NaAc), and NH4Cl. However, responses from the regenerated nerve in NaCl-restricted rats (40-120 d postsectioning) to NaCl and NaAc were reduced by as much as 30% compared to controls, indicating that regenerating taste receptors are influenced by environmental (dietary) factors. Responses to NH4Cl were normal; therefore, the effect appears specific to sodium salts. Surprisingly, in the same rats, NaCl responses from the contralateral, intact chorda tympani were up to 40% greater than controls. Thus, in the same rat, there was over a twofold difference in sodium responses between the right and left chorda tympani nerves. A study of the time course of the functional alterations in the intact nerve revealed that responses to NaCl were extremely low immediately following sectioning (about 20% of the normal response), and then increased monotonically during the following 50 d until relative response magnitudes became supersensitive. This function occurred even when the cut chorda tympani was prevented from reinnervating lingual epithelia, demonstrating that events related to regeneration do not play a role in the functional properties of the contralateral side of the tongue.(ABSTRACT TRUNCATED AT 250 WORDS)

Huntingtin Acts Non Cell-Autonomously on Hippocampal Neurogenesis and Controls Anxiety-Related Behaviors in Adult Mouse

PubMed Central

Pla, Patrick; Orvoen, Sophie; Benstaali, Caroline; Dodier, Sophie; Gardier, Alain M.; David, Denis J.; Humbert, Sandrine; Saudou, Frédéric

2013-01-01

Huntington’s disease (HD) is a fatal neurodegenerative disease, characterized by motor defects and psychiatric symptoms, including mood disorders such as anxiety and depression. HD is caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin (HTT) protein. The development and analysis of various mouse models that express pathogenic polyQ-HTT revealed a link between mutant HTT and the development of anxio-depressive behaviors and various hippocampal neurogenesis defects. However, it is unclear whether such phenotype is linked to alteration of HTT wild-type function in adults. Here, we report the analysis of a new mouse model in which HTT is inducibly deleted from adult mature cortical and hippocampal neurons using the CreERT2/Lox system. These mice present defects in both the survival and the dendritic arborization of hippocampal newborn neurons. Our data suggest that these non-cell autonomous effects are linked to defects in both BDNF transport and release upon HTT silencing in hippocampal neurons, and in BDNF/TrkB signaling. The controlled deletion of HTT also had anxiogenic-like effects. Our results implicate endogenous wild-type HTT in adult hippocampal neurogenesis and in the control of mood disorders. PMID:24019939

Enrichment and isolation of neurons from adult mouse brain for ex vivo analysis.

PubMed

Berl, Sabina; Karram, Khalad; Scheller, Anja; Jungblut, Melanie; Kirchhoff, Frank; Waisman, Ari

2017-05-01

Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases. Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads. Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation. Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression. These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS. Copyright © 2017 Elsevier B.V. All rights reserved.

FINE STRUCTURE OF CELLS ISOLATED FROM ADULT MOUSE LIVER

PubMed Central

Berry, M. N.; Simpson, F. O.

1962-01-01

Suspensions of isolated cells in various media were prepared from mouse liver which had been perfused via the portal vein with a buffered medium containing 0.40 M sucrose, and the cells were fixed with osmium tetroxide. Their fine structure was compared with that of cells from perfused and unperfused intact liver. Perfusion brought about some separation of the cells with little or no damage to cell membranes. When cells were dispersed in 0.40 M sucrose medium the plasma membranes partially broke down, and this disintegration was increased by transfer of the cells to media of lower osmolarity. This is presumed to account for the loss of permeability barriers which occurs in isolated liver cells. The mitochondria in cells of perfused liver and in isolated cells remained elongated, but the layers of many mitochondrial cristae became separated by clear spaces. When cells were transferred to a medium containing 0.20 M sucrose, the mitochondria swelled and became spherical, often with displacement of the swollen cristae to the periphery. In a medium containing 0.06 M sucrose and 0.08 M potassium chloride the outer chamber of many mitochondria became swollen with displacement of the mitochondrial body to one side to give a crescent-shaped appearance. These changes in mitochondrial morphology are discussed in relation to the metabolic activity of isolated liver cells. PMID:19866610

[Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

PubMed

Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

2017-01-01

Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

Deep-brain magnetic stimulation promotes adult hippocampal neurogenesis and alleviates stress-related behaviors in mouse models for neuropsychiatric disorders

PubMed Central

2014-01-01

Background Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood. Results Here we studied the effects of deep-brain magnetic stimulation to brain on the molecular and cellular level. We examined the adult hippocampal neurogenesis and hippocampal synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation treatment. We found that DMS promotes adult hippocampal neurogenesis significantly and facilitates the development of adult new-born neurons. Remarkably, DMS exerts anti-depression effects in the learned helplessness mouse model and rescues hippocampal long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates the stress response in a mouse model for Rett syndrome and prolongs the life span of these animals dramatically. Conclusions Deep-brain magnetic stimulation greatly facilitates adult hippocampal neurogenesis and maturation, also alleviates depression and stress-related responses in animal models. PMID:24512669

Ablation of Mouse Adult Neurogenesis Alters Olfactory Bulb Structure and Olfactory Fear Conditioning

PubMed Central

Valley, Matthew T.; Mullen, Tanner R.; Schultz, Lucy C.; Sagdullaev, Botir T.; Firestein, Stuart

2009-01-01

Adult neurogenesis replenishes olfactory bulb (OB) interneurons throughout the life of most mammals, yet during this constant flux it remains unclear how the OB maintains a constant structure and function. In the mouse OB, we investigated the dynamics of turnover and its impact on olfactory function by ablating adult neurogenesis with an x-ray lesion to the sub-ventricular zone (SVZ). Regardless of the magnitude of the lesion to the SVZ, we found no change in the survival of young adult born granule cells (GCs) born after the lesion, and a gradual decrease in the population of GCs born before the lesion. After a lesion producing a 96% reduction of incoming adult born GCs to the OB, we found a diminished behavioral fear response to conditioned odor cues but not to audio cues. Interestingly, despite this behavioral deficit and gradual anatomical changes, we found no electrophysiological changes in the GC population assayed in vivo through dendro-dendritic synaptic plasticity and odor-evoked local field potential oscillations. These data indicate that turnover in the granule cell layer is generally decoupled from the rate of adult neurogenesis, and that OB adult neurogenesis plays a role in a wide behavioral system extending beyond the OB. PMID:20582278

CD24-Positive Cells from Normal Adult Mouse Liver Are Hepatocyte Progenitor Cells

PubMed Central

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M.; Rao, Pulivarthi H.

2011-01-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45−, Ter119−) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes. PMID:21361791

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

PubMed

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M; Rao, Pulivarthi H; Darlington, Gretchen J

2011-12-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.

NIH mouse study finds gut microorganisms may determine cancer treatment outcome

Cancer.gov

An intact gut commensal microbiota, which is a population of microorganisms living in the intestine, is required for optimal response to cancer therapy, according to a mouse study by scientists at the National Cancer Institute (NCI)

Function of GATA Factors in the Adult Mouse Liver

PubMed Central

Zheng, Rena; Rebolledo-Jaramillo, Boris; Zong, Yiwei; Wang, Liqing; Russo, Pierre; Hancock, Wayne; Stanger, Ben Z.; Hardison, Ross C.; Blobel, Gerd A.

2013-01-01

GATA transcription factors and their Friend of Gata (FOG) cofactors control the development of diverse tissues. GATA4 and GATA6 are essential for the expansion of the embryonic liver bud, but their expression patterns and functions in the adult liver are unclear. We characterized the expression of GATA and FOG factors in whole mouse liver and purified hepatocytes. GATA4, GATA6, and FOG1 are the most prominently expressed family members in whole liver and hepatocytes. GATA4 chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) identified 4409 occupied sites, associated with genes enriched in ontologies related to liver function, including lipid and glucose metabolism. However, hepatocyte-specific excision of Gata4 had little impact on gross liver architecture and function, even under conditions of regenerative stress, and, despite the large number of GATA4 occupied genes, resulted in relatively few changes in gene expression. To address possible redundancy between GATA4 and GATA6, both factors were conditionally excised. Surprisingly, combined Gata4,6 loss did not exacerbate the phenotype resulting from Gata4 loss alone. This points to the presence of an unusually robust transcriptional network in adult hepatocytes that ensures the maintenance of liver function. PMID:24367609

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

PubMed

Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

2011-06-01

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

PubMed Central

Kilpatrick, Lauren A.; Li, Qian; Yang, John; Goddard, John C; Fekete, Donna M.; Lang, Hainan

2010-01-01

Murine models are ideal for studying cochlear gene transfer as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, due to its small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAV) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear and allows for near-complete preservation of low and middle frequency hearing. In the present study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6, and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus (CMV) promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells (IHCs) were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness. PMID:21209625

Whole-Mount Adult Ear Skin Imaging Reveals Defective Neuro-Vascular Branching Morphogenesis in Obese and Type 2 Diabetic Mouse Models.

PubMed

Yamazaki, Tomoko; Li, Wenling; Yang, Ling; Li, Ping; Cao, Haiming; Motegi, Sei-Ichiro; Udey, Mark C; Bernhard, Elise; Nakamura, Takahisa; Mukouyama, Yoh-Suke

2018-01-11

Obesity and type 2 diabetes are frequently associated with peripheral neuropathy. Though there are multiple methods for diagnosis and analysis of morphological changes of peripheral nerves and blood vessels, three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis with an anatomically recognizable branching morphogenesis and patterning. Here we established a novel technique for whole-mount imaging of adult mouse ear skin to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels. Whole-mount immunostaining of adult mouse ear skin showed that peripheral sensory and sympathetic nerves align with large-diameter blood vessels. Diet-induced obesity (DIO) mice exhibit defective vascular smooth muscle cells (VSMCs) coverage, while there is no significant change in the amount of peripheral nerves. The leptin receptor-deficient db/db mice, a severe obese and type 2 diabetic mouse model, exhibit defective VSMC coverage and a large increase in the amount of smaller-diameter nerve bundles with myelin sheath and unmyelinated nerve fibers. Interestingly, an increase in the amount of myeloid immune cells was observed in the DIO but not db/db mouse skin. These data suggest that our whole-mount imaging method enables us to investigate the neuro-vascular and neuro-immune phenotypes in the animal models of obesity and diabetes.

Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres.

PubMed

Hamdi, M M; Mutungi, G

2010-02-01

It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.

PubMed

Zemmar, Ajmal; Chen, Chia-Chien; Weinmann, Oliver; Kast, Brigitt; Vajda, Flora; Bozeman, James; Isaad, Noel; Zuo, Yi; Schwab, Martin E

2018-06-01

Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.

In situ assessment of shortening and lengthening contractile properties of hind limb ankle flexors in intact mice.

PubMed

Gorselink, M; Drost, M R; de Louw, J; Willems, P J; Hesselink, M K; Dekkers, E C; Rosielle, N; van der Vusse, G J

2001-05-01

The availability of animal models with disrupted genes has increased the need for small-scale measurement devices. Recently, we developed an experimental device to assess in situ mechanical properties of isometric contractions of intact muscle complexes of the mouse. Although this apparatus provides valuable information on muscle mechanical performance, it is not appropriate for determining contractile properties during shortening and lengthening contractions. In the present study we therefore developed and evaluated an experimental apparatus for assessment of shortening and lengthening contractile properties of intact plantar and dorsal flexors of the mouse. The current through a custom-built, low-inertia servomotor was measured to assess contractile muscular torque ranging from -50 to mN.m. Evaluation of the fixation procedure of the animal to the apparatus via 3-D monitoring of the muscle-tendon complex length showed that the additional shortening in length due to a contraction with maximal torque output has only minor effects on the measured torque. Furthermore, misalignment of the axis of rotation of the apparatus relative to the axis of rotation in the ankle joint, i.e. eccentricity, during a routine experiment was estimated to be less than 1.0 mm and hence did not influence the measured torque output under our experimental conditions. Peak power per unit muscle mass (mean +/- SD) of intact dorsal and plantar flexors was 0.27 +/- 0.02 and 0.19 +/- 0.03 W.g-1, respectively. The angular velocity at maximal peak power generated by the dorsal flexor complex and the plantar flexor complex was 1100 +/- 190 and 700 +/- 90 degrees.s-1, respectively.

Astrocyte activation and wound healing in intact-skull mouse after focal brain injury.

PubMed

Suzuki, Takayuki; Sakata, Honami; Kato, Chiaki; Connor, John A; Morita, Mitsuhiro

2012-12-01

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery. © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

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

Bleil, J.D.; Wassarman, P.M.

1986-04-01

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetratemore » the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.« less

Neural stem/progenitor cell properties of glial cells in the adult mouse auditory nerve

PubMed Central

Lang, Hainan; Xing, Yazhi; Brown, LaShardai N.; Samuvel, Devadoss J.; Panganiban, Clarisse H.; Havens, Luke T.; Balasubramanian, Sundaravadivel; Wegner, Michael; Krug, Edward L.; Barth, Jeremy L.

2015-01-01

The auditory nerve is the primary conveyor of hearing information from sensory hair cells to the brain. It has been believed that loss of the auditory nerve is irreversible in the adult mammalian ear, resulting in sensorineural hearing loss. We examined the regenerative potential of the auditory nerve in a mouse model of auditory neuropathy. Following neuronal degeneration, quiescent glial cells converted to an activated state showing a decrease in nuclear chromatin condensation, altered histone deacetylase expression and up-regulation of numerous genes associated with neurogenesis or development. Neurosphere formation assays showed that adult auditory nerves contain neural stem/progenitor cells (NSPs) that were within a Sox2-positive glial population. Production of neurospheres from auditory nerve cells was stimulated by acute neuronal injury and hypoxic conditioning. These results demonstrate that a subset of glial cells in the adult auditory nerve exhibit several characteristics of NSPs and are therefore potential targets for promoting auditory nerve regeneration. PMID:26307538

Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras

PubMed Central

Keighren, Margaret A.; Flockhart, Jean H.

2016-01-01

ABSTRACT The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1−/− null mouse embryos die but a previous study showed that some homozygous Gpi1−/− null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1−/−↔Gpi1c/c chimaera with functional Gpi1−/− null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1−/− null cells in adult Gpi1−/−↔Gpi1c/c chimaeras and determine if Gpi1−/− null germ cells are functional. Analysis of adult Gpi1−/−↔Gpi1c/c chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1−/− null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1−/− null oocytes in one female Gpi1−/−↔Gpi1c/c chimaera were functional and provided preliminary evidence that one male putative Gpi1−/−↔Gpi1c/c chimaera produced functional spermatozoa from homozygous Gpi1−/− null germ cells. Although the male chimaera was almost certainly Gpi1−/−↔Gpi1c/c, this part of the study is considered preliminary because only blood was typed for GPI. Gpi1−/− null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1−/− null germ cells, it successfully identified functional Gpi1−/− null oocytes and revealed that some Gpi1−/− null cells could survive in many adult tissues. PMID:27103217

Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

PubMed Central

Carreira, Vinicius S.; Fan, Yunxia; Kurita, Hisaka; Wang, Qin; Ko, Chia-I; Naticchioni, Mindi; Jiang, Min; Koch, Sheryl; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Xia, Ying; Rubinstein, Jack; Puga, Alvaro

2015-01-01

The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr -/- and in utero TCDD-exposed Ahr +/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr -/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease. PMID:26555816

C/EBPα and C/EBPβ Are Required for Sebocyte Differentiation and Stratified Squamous Differentiation in Adult Mouse Skin

PubMed Central

House, John S.; Zhu, Songyun; Ranjan, Rakesh; Linder, Keith; Smart, Robert C.

2010-01-01

C/EBPα and C/EBPβ are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPα and C/EBPβ in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPα or C/EBPβ alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPα and C/EBPβ in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPα and C/EBPβ in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPα and C/EBPβ are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal. PMID:20352127

C/EBPalpha and C/EBPbeta are required for Sebocyte differentiation and stratified squamous differentiation in adult mouse skin.

PubMed

House, John S; Zhu, Songyun; Ranjan, Rakesh; Linder, Keith; Smart, Robert C

2010-03-23

C/EBPalpha and C/EBPbeta are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPalpha and C/EBPbeta in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPalpha or C/EBPbeta alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPalpha and C/EBPbeta in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPalpha and C/EBPbeta in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3) and melanocortin 5 receptor (MC5R), two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPalpha and C/EBPbeta are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal.

Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

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

Sun, Li; Wu, Zhou; Baba, Masashi

Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, themore » understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in

Intracellular pH change does not accompany egg activation in the mouse.

PubMed

Phillips, K P; Baltz, J M

1996-09-01

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi). We measured pHi in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH, was 6.96 +/- 0.004 (+/- SEM) in GV-intact oocytes, 7.00 +/- 0.01 in ovulated, unfertilized eggs, and 7.02 +/- 0.01 in fertilized zygotes, indicating no sustained changes in pHi after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pHi occur shortly after egg activation, mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS); no significant change in pHi followed ethanol activation. Since increased Na+/H+ antiporter activity is responsible for pHi increase in the sea urchin, pHi was measured in the absence of added bicarbonate or CO2 (a condition under which the antiporter would be the only major pHi regulatory mechanism able to operate, since the others were bicarbonate-dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na+/H+ antiporter was activated. There was no physiologically significant difference in pHi after GVBD or fertilization, when pHi was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation. Thus, a change in pHi is not a feature of egg activation in the mouse.

Language of administration and neuropsychological test performance in neurologically intact Hispanic American bilingual adults.

PubMed

Gasquoine, Philip Gerard; Croyle, Kristin L; Cavazos-Gonzalez, Cynthia; Sandoval, Omar

2007-11-01

This study compared the performance of Hispanic American bilingual adults on Spanish and English language versions of a neuropsychological test battery. Language achievement test scores were used to divide 36 bilingual, neurologically intact, Hispanic Americans from south Texas into Spanish-dominant, balanced, and English-dominant bilingual groups. They were administered the eight subtests of the Bateria Neuropsicologica and the Matrix Reasoning subtest of the WAIS-III in Spanish and English. Half the participants were tested in Spanish first. Balanced bilinguals showed no significant differences in test scores between Spanish and English language administrations. Spanish and/or English dominant bilinguals showed significant effects of language of administration on tests with higher language compared to visual perceptual weighting (Woodcock-Munoz Language Survey-Revised, Letter Fluency, Story Memory, and Stroop Color and Word Test). Scores on tests with higher visual-perceptual weighting (Matrix Reasoning, Figure Memory, Wisconsin Card Sorting Test, and Spatial Span), were not significantly affected by language of administration, nor were scores on the Spanish/California Verbal Learning Test, and Digit Span. A problem was encountered in comparing false positive rates in each language, as Spanish norms fell below English norms, resulting in a much higher false positive rate in English across all bilingual groupings. Use of a comparison standard (picture vocabulary score) reduced false positive rates in both languages, but the higher false positive rate in English persisted.

Comparison of allergenicity and immunogenicity of an intact allergen vaccine and commercially available allergoid products for birch pollen immunotherapy.

PubMed

Lund, L; Henmar, H; Würtzen, P A; Lund, G; Hjortskov, N; Larsen, J N

2007-04-01

Specific immunotherapy with intact allergen vaccine is a well-documented treatment for allergic diseases. Different vaccine formulations are currently commercially available, the active ingredient either being intact allergens or chemically modified allergoids. The rationale behind allergoids is to decrease allergenicity while maintaining immunogenicity. However, data from the German health authorities based on reporting of adverse events over a 10-year period did not indicate increased safety of allergoids over intact allergens. The objective of this study was to investigate the effect of chemical modification on allergenicity and immunogenicity comparing four commercial allergoid products for birch pollen immunotherapy with an intact allergen vaccine. Solid-phase IgE inhibition and histamine release assays were selected as model systems for allergenicity, and a combination of human T cell proliferation and IgG titres following mouse immunizations were used to address the immunogenicity of the intact allergen vaccine and the four allergoids. In all assays, the products were normalized with respect to the manufacturer's recommended maintenance dose. IgE inhibition experiments showed a change in epitope composition comparing intact allergen vaccine with allergoid. One allergoid product induced enhanced histamine release compared to the intact allergens, while the other three allergoids showed reduced release. Standard T cell stimulation assays using lines from allergic patients showed a reduced response for all allergoids compared with the intact allergen vaccine regardless of the cell type used for antigen presentation. All allergoids showed reduced capacity to induce allergen-specific IgG responses in mice. While some allergoids were associated with reduced allergenicity, a clear reduction in immunogenicity was observed for all allergoid products compared with the intact allergen vaccine, and the commercial allergoids tested therefore do not fulfil the allergoid

Retinal lesions induce fast intrinsic cortical plasticity in adult mouse visual system.

PubMed

Smolders, Katrien; Vreysen, Samme; Laramée, Marie-Eve; Cuyvers, Annemie; Hu, Tjing-Tjing; Van Brussel, Leen; Eysel, Ulf T; Nys, Julie; Arckens, Lutgarde

2016-09-01

Neuronal activity plays an important role in the development and structural-functional maintenance of the brain as well as in its life-long plastic response to changes in sensory stimulation. We characterized the impact of unilateral 15° laser lesions in the temporal lower visual field of the retina, on visually driven neuronal activity in the afferent visual pathway of adult mice using in situ hybridization for the activity reporter gene zif268. In the first days post-lesion, we detected a discrete zone of reduced zif268 expression in the contralateral hemisphere, spanning the border between the monocular segment of the primary visual cortex (V1) with extrastriate visual area V2M. We could not detect a clear lesion projection zone (LPZ) in areas lateral to V1 whereas medial to V2M, agranular and granular retrosplenial cortex showed decreased zif268 levels over their full extent. All affected areas displayed a return to normal zif268 levels, and this was faster in higher order visual areas than in V1. The lesion did, however, induce a permanent LPZ in the retinorecipient layers of the superior colliculus. We identified a retinotopy-based intrinsic capacity of adult mouse visual cortex to recover from restricted vision loss, with recovery speed reflecting the areal cortical magnification factor. Our observations predict incomplete visual field representations for areas lateral to V1 vs. lack of retinotopic organization for areas medial to V2M. The validation of this mouse model paves the way for future interrogations of cortical region- and cell-type-specific contributions to functional recovery, up to microcircuit level. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Comparison of Intact PTH and Bio-Intact PTH Assays Among Non-Dialysis Dependent Chronic Kidney Disease Patients.

PubMed

Einbinder, Yael; Benchetrit, Sydney; Golan, Eliezer; Zitman-Gal, Tali

2017-09-01

The third-generation bio-intact parathyroid hormone (PTH) (1-84) assay was designed to overcome problems associated with the detection of C-terminal fragments by the second-generation intact PTH assay. The two assays have been compared primarily among dialysis populations. The present study evaluated the correlations and differences between these two PTH assays among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis. Blood samples were collected from 98 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation - PTH intact-STAT and third-generation bio-intact 1-84 PTH assays. Other serum biomarkers of bone mineral disorders were also assessed. CKD stage was calculated by using the CKD-Epidemiology Collaboration (EPI) formula. Serum bio-intact PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r=0.963, P<0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and bio-intact PTH) positively correlated with urea (r=0.523, r=0.504; P=0.002, respectively), phosphorus (r=0.532, r=0.521; P<0.0001, respectively) and negatively correlated with blood calcium (r=-0.435, r=-0.476; P<0.0001, respectively), 25(OH) vitamin D, (r=-0.319, r=-0.353; respectively, P<0.0001) and the estimated glomerular filtration rate (r=-0.717, r=-0.688; P<0.0001, respectively). Among patients with CKD stages 3 to 5 not on dialysis, the bio-intact PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders with bone histomorphometric findings are needed to determine whether bio-intact PTH assay results are better surrogate markers in these early stages of CKD. © The Korean Society for Laboratory Medicine

Recollections of Parent Characteristics and Attachment Patterns for College Women of Intact vs. Non-Intact Families

ERIC Educational Resources Information Center

Kilmann, Peter R.; Carranza, Laura V.; Vendemia, Jennifer M. C.

2006-01-01

This study contrasted offsprings' attachment patterns and recollections of parent characteristics in two college samples: 147 females from intact biological parents and 157 females of parental divorce. Secure females from intact or non-intact families rated parents positively, while insecure females rated parents as absent, distant, and demanding.…

Localization and regulation of PML bodies in the adult mouse brain.

PubMed

Hall, Małgorzata H; Magalska, Adriana; Malinowska, Monika; Ruszczycki, Błażej; Czaban, Iwona; Patel, Satyam; Ambrożek-Latecka, Magdalena; Zołocińska, Ewa; Broszkiewicz, Hanna; Parobczak, Kamil; Nair, Rajeevkumar R; Rylski, Marcin; Pawlak, Robert; Bramham, Clive R; Wilczyński, Grzegorz M

2016-06-01

PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.

Wnt3 and Gata4 regulate axon regeneration in adult mouse DRG neurons.

PubMed

Duan, Run-Shan; Liu, Pei-Pei; Xi, Feng; Wang, Wei-Hua; Tang, Gang-Bin; Wang, Rui-Ying; Saijilafu; Liu, Chang-Mei

2018-05-05

Neurons in the adult central nervous system (CNS) have a poor intrinsic axon growth potential after injury, but the underlying mechanisms are largely unknown. Wingless-related mouse mammary tumor virus integration site (WNT) family members regulate neural stem cell proliferation, axon tract and forebrain development in the nervous system. Here we report that Wnt3 is an important modulator of axon regeneration. Downregulation or overexpression of Wnt3 in adult dorsal root ganglion (DRG) neurons enhances or inhibits their axon regeneration ability respectively in vitro and in vivo. Especially, we show that Wnt3 modulates axon regeneration by repressing mRNA translation of the important transcription factor Gata4 via binding to the three prime untranslated region (3'UTR). Downregulation of Gata4 could restore the phenotype exhibited by Wnt3 downregulation in DRG neurons. Taken together, these data indicate that Wnt3 is a key intrinsic regulator of axon growth ability of the nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of N-methyl-D-aspartate-evoked taurine release in the developing and adult mouse hippocampus.

PubMed

Saransaari, P; Oja, S S

2003-01-01

Taurine is an inhibitory amino acid acting as an osmoregulator and neuroromodulator in the brain, with neuroprotective properties. The ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) greatly potentiates taurine release from brain preparations in both normal and ischemic conditions, the effect being particularly marked in the developing hippocampus. We now characterized the regulation of NMDA-stimulated taurine release from hippocampal slices from adult (3-month-old) and developing (7-day-old) mouse using a superfusion system. The NMDA-stimulated taurine release was receptor-mediated in both adult and developing mouse hippocampus. In adults, only NO-generating compounds, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and hydroxylamine reduced the release, as did also NO synthase inhibitors, 7-nitroindazole and nitroarginine, indicating that the release is mediated by the NO/cGMP pathway. On the other hand, the regulation of the NMDA-evoked taurine release proved to be somewhat complex in the immature hippocampus. It was not affected by the NOergic compounds, but enhanced by the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate and adenosine receptor A(1) agonists, N(6)-cyclohexyladenosine and R(-)N(6)-(2-phenylisopropyl)adenosine in a receptor-mediated manner. The activation of both ionotropic 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and metabotropic glutamate group I receptors also enhanced the evoked release. The NMDA-receptor-stimulated taurine release could be a part of the neuroprotective properties of taurine, being important particularly under cell-damaging conditions in the developing hippocampus and hence preventing excitotoxicity.

In Amnio MRI of Mouse Embryos

PubMed Central

Roberts, Thomas A.; Norris, Francesca C.; Carnaghan, Helen; Savery, Dawn; Wells, Jack A.; Siow, Bernard; Scambler, Peter J.; Pierro, Agostino; De Coppi, Paolo; Eaton, Simon; Lythgoe, Mark F.

2014-01-01

Mouse embryo imaging is conventionally carried out on ex vivo embryos excised from the amniotic sac, omitting vital structures and abnormalities external to the body. Here, we present an in amnio MR imaging methodology in which the mouse embryo is retained in the amniotic sac and demonstrate how important embryonic structures can be visualised in 3D with high spatial resolution (100 µm/px). To illustrate the utility of in amnio imaging, we subsequently apply the technique to examine abnormal mouse embryos with abdominal wall defects. Mouse embryos at E17.5 were imaged and compared, including three normal phenotype embryos, an abnormal embryo with a clear exomphalos defect, and one with a suspected gastroschisis phenotype. Embryos were excised from the mother ensuring the amnion remained intact and stereo microscopy was performed. Embryos were next embedded in agarose for 3D, high resolution MRI on a 9.4T scanner. Identification of the abnormal embryo phenotypes was not possible using stereo microscopy or conventional ex vivo MRI. Using in amnio MRI, we determined that the abnormal embryos had an exomphalos phenotype with varying severities. In amnio MRI is ideally suited to investigate the complex relationship between embryo and amnion, together with screening for other abnormalities located outside of the mouse embryo, providing a valuable complement to histology and existing imaging methods available to the phenotyping community. PMID:25330230

Coenzyme Q(1) as a probe for mitochondrial complex I activity in the intact perfused hyperoxia-exposed wild-type and Nqo1-null mouse lung.

PubMed

Bongard, Robert D; Myers, Charles R; Lindemer, Brian J; Baumgardt, Shelley; Gonzalez, Frank J; Merker, Marilyn P

2012-05-01

Previous studies showed that coenzyme Q(1) (CoQ(1)) reduction on passage through the rat pulmonary circulation was catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex I, but that NQO1 genotype was not a factor in CoQ(1) reduction on passage through the mouse lung. The aim of the present study was to evaluate the complex I contribution to CoQ(1) reduction in the isolated perfused wild-type (NQO1(+/+)) and Nqo1-null (NQO1(-)/(-)) mouse lung. CoQ(1) reduction was measured as the steady-state pulmonary venous CoQ(1) hydroquinone (CoQ(1)H(2)) efflux rate during infusion of CoQ(1) into the pulmonary arterial inflow. CoQ(1)H(2) efflux rates during infusion of 50 μM CoQ(1) were not significantly different for NQO1(+/+) and NQO1(-/-) lungs (0.80 ± 0.03 and 0.68 ± 0.07 μmol·min(-1)·g lung dry wt(-1), respectively, P > 0.05). The mitochondrial complex I inhibitor rotenone depressed CoQ(1)H(2) efflux rates for both genotypes (0.19 ± 0.08 and 0.08 ± 0.04 μmol·min(-1)·g lung dry wt(-1) for NQO1(+/+) and NQO1(-/-), respectively, P < 0.05). Exposure of mice to 100% O(2) for 48 h also depressed CoQ(1)H(2) efflux rates in NQO1(+/+) and NQO1(-/-) lungs (0.43 ± 0.03 and 0.11 ± 0.04 μmol·min(-1)·g lung dry wt(-1), respectively, P < 0.05 by ANOVA). The impact of rotenone or hyperoxia on CoQ(1) redox metabolism could not be attributed to effects on lung wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total venous effluent CoQ(1) recoveries, the latter measured by spectrophotometry or mass spectrometry. Complex I activity in mitochondria-enriched lung fractions was depressed in hyperoxia-exposed lungs for both genotypes. This study provides new evidence for the potential utility of CoQ(1) as a nondestructive indicator of the impact of pharmacological or pathological exposures on complex I activity in the intact perfused mouse lung.

Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus.

PubMed

Chachlaki, Konstantina; Malone, Samuel A; Qualls-Creekmore, Emily; Hrabovszky, Erik; Münzberg, Heike; Giacobini, Paolo; Ango, Fabrice; Prevot, Vincent

2017-10-15

Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP Vglut2 , EYFP Vgat , and GFP Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes. © 2017 Wiley Periodicals, Inc.

An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris

PubMed Central

Schulze, Katja; Galichet, Arnaud; Sayar, Beyza S.; Scothern, Anthea; Howald, Denise; Zymann, Hillard; Siffert, Myriam; Zenhäusern, Denise; Bolli, Reinhard; Koch, Peter J.; Garrod, David; Suter, Maja M.; Müller, Eliane J.

2011-01-01

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may play a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice which have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we here developed a model with passive transfer of the monospecific pathogenic Dsg3 antibody AK23 into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies. PMID:21956125

pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

PubMed

Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

2017-09-05

The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries

PubMed Central

Woods, Dori C; Tilly, Jonathan L

2017-01-01

Accruing evidence indicates that production of new oocytes (oogenesis) and their enclosure by somatic cells (folliculogenesis) are processes not limited to the perinatal period in mammals. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life. The recent purification of mitotically active oocyte progenitor cells, termed female germline stem cells (fGSCs) or oogonial stem cells (OSCs), from mouse and human ovaries opens up new avenues for research into the biology and clinical utility of these cells. Here we detail methods for the isolation of mouse and human OSCs from adult ovarian tissue, cultivation of the cells after purification, and characterization of the cells before and after ex vivo expansion. The latter methods include analysis of germ cell–specific markers and in vitro oogenesis, as well as the use of intraovarian transplantation to test the oocyte-forming potential of OSCs in vivo. PMID:23598447

Intact skull chronic windows for mesoscopic wide-field imaging in awake mice

PubMed Central

Silasi, Gergely; Xiao, Dongsheng; Vanni, Matthieu P.; Chen, Andrew C. N.; Murphy, Timothy H.

2016-01-01

Background Craniotomy-based window implants are commonly used for microscopic imaging, in head-fixed rodents, however their field of view is typically small and incompatible with mesoscopic functional mapping of cortex. New Method We describe a reproducible and simple procedure for chronic through-bone wide-field imaging in awake head-fixed mice providing stable optical access for chronic imaging over large areas of the cortex for months. Results The preparation is produced by applying clear-drying dental cement to the intact mouse skull, followed by a glass coverslip to create a partially transparent imaging surface. Surgery time takes about 30 minutes. A single set-screw provides a stable means of attachment for mesoscale assessment without obscuring the cortical field of view. Comparison with Existing Methods We demonstrate the utility of this method by showing seed-pixel functional connectivity maps generated from spontaneous cortical activity of GCAMP6 signals in both awake and anesthetized mice. Conclusions We propose that the intact skull preparation described here may be used for most longitudinal studies that do not require micron scale resolution and where cortical neural or vascular signals are recorded with intrinsic sensors. PMID:27102043

Anthocyanins protect against LPS-induced oxidative stress-mediated neuroinflammation and neurodegeneration in the adult mouse cortex.

PubMed

Khan, Muhammad Sohail; Ali, Tahir; Kim, Min Woo; Jo, Myeung Hoon; Jo, Min Gi; Badshah, Haroon; Kim, Myeong Ok

2016-11-01

Several studies provide evidence that reactive oxygen species (ROS) are key mediators of various neurological disorders. Anthocyanins are polyphenolic compounds and are well known for their anti-oxidant and neuroprotective effects. In this study, we investigated the neuroprotective effects of anthocyanins (extracted from black soybean) against lipopolysaccharide (LPS)-induced ROS-mediated neuroinflammation and neurodegeneration in the adult mouse cortex. Intraperitoneal injection of LPS (250 μg/kg) for 7 days triggers elevated ROS and oxidative stress, which induces neuroinflammation and neurodegeneration in the adult mouse cortex. Treatment with 24 mg/kg/day of anthocyanins for 14 days in LPS-injected mice (7 days before and 7 days co-treated with LPS) attenuated elevated ROS and oxidative stress compared to mice that received LPS-injection alone. The immunoblotting results showed that anthocyanins reduced the level of the oxidative stress kinase phospho-c-Jun N-terminal Kinase 1 (p-JNK). The immunoblotting and morphological results showed that anthocyanins treatment significantly reduced LPS-induced-ROS-mediated neuroinflammation through inhibition of various inflammatory mediators, such as IL-1β, TNF-α and the transcription factor NF- k B. Anthocyanins treatment also reduced activated astrocytes and microglia in the cortex of LPS-injected mice, as indicated by reductions in GFAP and Iba-1, respectively. Anthocyanins also prevent overexpression of various apoptotic markers, i.e., Bax, cytosolic cytochrome C, cleaved caspase-3 and PARP-1. Immunohistochemical fluoro-jade B (FJB) and Nissl staining indicated that anthocyanins prevent LPS-induced neurodegeneration in the mouse cortex. Our results suggest that dietary flavonoids, such as anthocyanins, have antioxidant and neuroprotective activities that could be beneficial to various neurological disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy

PubMed Central

Henmar, H; Lund, G; Lund, L; Petersen, A; Würtzen, P A

2008-01-01

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients. PMID:18647321

Allergenicity, immunogenicity and dose-relationship of three intact allergen vaccines and four allergoid vaccines for subcutaneous grass pollen immunotherapy.

PubMed

Henmar, H; Lund, G; Lund, L; Petersen, A; Würtzen, P A

2008-09-01

Different vaccines containing intact allergens or chemically modified allergoids as active ingredients are commercially available for specific immunotherapy. Allergoids are claimed to have decreased allergenicity without loss of immunogenicity and this is stated to allow administration of high allergoid doses. We compared the allergenicity and immunogenicity of four commercially available chemically modified grass pollen allergoid products with three commercially available intact grass pollen allergen vaccines. The allergenicity was investigated with immunoglobulin (Ig)E-inhibition and basophil activation assays. Human T cell proliferation and specific IgG-titres following mouse immunizations were used to address immunogenicity. Furthermore, intact allergen vaccines with different contents of active ingredients were selected to study the influence of the allergen dose. In general, a lower allergenicity for allergen vaccines was clearly linked to a reduced immunogenicity. Compared with the vaccine with the highest amount of intact allergen, the allergoids caused reduced basophil activation as well as diminished immunogenicity demonstrated by reduced T cell activation and/or reduced induction of murine grass-specific IgG antibodies. Interestingly, intact allergen vaccines with lower content of active ingredient exhibited similarly reduced allergenicity, while immunogenicity was still higher or equal to that of allergoids. The low allergenicity observed for some allergoids was inherently linked to a significantly lower immunogenic response questioning the rationale behind the chemical modification into allergoids. In addition, the linkage between allergenicity, immunogenicity and dose found for intact allergen vaccines and the immunogen as well as allergenic immune responses observed for allergoids suggest that the modified allergen vaccines do not contain high doses of immunologically active ingredients.

Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

PubMed Central

Mahalingam, Sharada; Gao, Liying; Gonnering, Marni; Helferich, William; Flaws, Jodi A.

2016-01-01

Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral follicles isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. PMID:26876617

Microinjection of Follicle-Enclosed Mouse Oocytes

NASA Astrophysics Data System (ADS)

Jaffe, Laurinda A.; Norris, Rachael P.; Freudzon, Marina; Ratzan, William J.; Mehlmann, Lisa M.

The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

Comprehensive interactome of Otx2 in the adult mouse neural retina.

PubMed

Fant, Bruno; Samuel, Alexander; Audebert, Stéphane; Couzon, Agnès; El Nagar, Salsabiel; Billon, Nathalie; Lamonerie, Thomas

2015-11-01

The Otx2 homeodomain transcription factor exerts multiple functions in specific developmental contexts, probably through the regulation of different sets of genes. Protein partners of Otx2 have been shown to modulate its activity. Therefore, the Otx2 interactome may play a key role in selecting a precise target-gene repertoire, hence determining its function in a specific tissue. To address the nature of Otx2 interactome, we generated a new recombinant Otx2(CTAP-tag) mouse line, designed for protein complexes purification. We validated this mouse line by establishing the Otx2 interactome in the adult neural retina. In this tissue, Otx2 is thought to have overlapping function with its paralog Crx. Our analysis revealed that, in contrary to Crx, Otx2 did not develop interactions with proteins that are known to regulate phototransduction genes but showed specific partnership with factors associated with retinal development. The relationship between Otx2 and Crx in the neural retina should therefore be considered as complementarity rather than redundancy. Furthermore, study of the Otx2 interactome revealed strong associations with RNA processing and translation machineries, suggesting unexpected roles for Otx2 in the regulation of selected target genes all along the transcription/translation pathway. The Otx2(CTAP-tag) line, therefore, appears suitable for a systematic approach to Otx2 protein-protein interactions. genesis 53:685-694, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Positive Youth Development, Life Satisfaction, and Problem Behaviors of Adolescents in Intact and Non-Intact Families in Hong Kong

PubMed Central

Shek, Daniel T. L.; Leung, Hildie

2013-01-01

This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families. PMID:24400264

Positive youth development, life satisfaction, and problem behaviors of adolescents in intact and non-intact families in Hong Kong.

PubMed

Shek, Daniel T L; Leung, Hildie

2013-01-01

This study investigated whether Chinese adolescents living in intact and non-intact families differed in their positive development, life satisfaction, and risk behavior. A total of 3,328 Secondary 1 students responded to measures of positive youth development (such as resilience and psychosocial competencies), life satisfaction, and risk behavior (substance abuse, delinquency, Internet addiction, consumption of pornographic materials, self-harm, and behavioral intention to engage in problem behavior). Findings revealed that adolescents growing up in intact families reported higher levels of positive developmental outcomes and life satisfaction as compared with adolescents from non-intact families. Adolescents in non-intact families also reported higher levels of risk behaviors than those growing up in intact families.

SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

PubMed Central

Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.

2011-01-01

SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168

Exploratory investigations of hypervelocity intact capture spectroscopy

NASA Technical Reports Server (NTRS)

Tsou, P.; Griffiths, D. J.

1993-01-01

The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

Developmental amnesia: a new pattern of dissociation with intact episodic memory.

PubMed

Temple, Christine M; Richardson, Paul

2004-01-01

A case of developmental amnesia is reported for a child, CL, of normal intelligence, who has intact episodic memory but impaired semantic memory for both semantic knowledge of facts and semantic knowledge of words, including general world knowledge, knowledge of word meanings and superordinate knowledge of words. In contrast to the deficits in semantic memory, there are no impairments in episodic memory for verbal or visual material, assessed by recall or recognition. Lexical decision was also intact, indicating impairment in semantic knowledge of vocabulary rather than absence of lexical representations. The case forms a double dissociation to the cases of Vargha-Khadem et al. [Science 277 (1997) 376; Episodic memory: new directions in research (2002) 153]; Gadian et al. [Brain 123 (2000) 499] for whom semantic memory was intact but episodic memory was impaired. This double dissociation suggests that semantic memory and episodic memory have the capacity to develop separately and supports models of modularity within memory development and a functional architecture for the developmental disorders within which there is residual normality rather than pervasive abnormality. Knowledge of arithmetical facts is also spared for CL, consistent with adult studies arguing for numeracy knowledge distinct from other semantics. Reading was characterised by difficulty with irregular words and homophones but intact reading of nonwords. CL has surface dyslexia with poor lexico-semantic reading skills but good phonological reading skills. The case was identified following screening from a population of normal schoolchildren suggesting that developmental amnesias may be more pervasive than has been recognised previously.

Intact glycopeptide characterization using mass spectrometry.

PubMed

Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

2016-05-01

Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.

Area-Specific Regulation of Quiescent Neural Stem Cells by Notch3 in the Adult Mouse Subependymal Zone.

PubMed

Kawai, Hiroki; Kawaguchi, Daichi; Kuebrich, Benjamin D; Kitamoto, Takeo; Yamaguchi, Masahiro; Gotoh, Yukiko; Furutachi, Shohei

2017-12-06

In the adult mammalian brain, neural stem cells (NSCs) generate new neurons throughout the mammal's lifetime. The balance between quiescence and active cell division among NSCs is crucial in producing appropriate numbers of neurons while maintaining the stem cell pool for a long period. The Notch signaling pathway plays a central role in both maintaining quiescent NSCs (qNSCs) and promoting cell division of active NSCs (aNSCs), although no one knows how this pathway regulates these apparently opposite functions. Notch1 has been shown to promote proliferation of aNSCs without affecting qNSCs in the adult mouse subependymal zone (SEZ). In this study, we found that Notch3 is expressed to a higher extent in qNSCs than in aNSCs while Notch1 is preferentially expressed in aNSCs and transit-amplifying progenitors in the adult mouse SEZ. Furthermore, Notch3 is selectively expressed in the lateral and ventral walls of the SEZ. Knockdown of Notch3 in the lateral wall of the adult SEZ increased the division of NSCs. Moreover, deletion of the Notch3 gene resulted in significant reduction of qNSCs specifically in the lateral and ventral walls, compared with the medial and dorsal walls, of the lateral ventricles. Notch3 deletion also reduced the number of qNSCs activated after antimitotic cytosine β-D-arabinofuranoside (Ara-C) treatment. Importantly, Notch3 deletion preferentially reduced specific subtypes of newborn neurons in the olfactory bulb derived from the lateral walls of the SEZ. These results indicate that Notch isoforms differentially control the quiescent and proliferative steps of adult SEZ NSCs in a domain-specific manner. SIGNIFICANCE STATEMENT In the adult mammalian brain, the subependymal zone (SEZ) of the lateral ventricles is the largest neurogenic niche, where neural stem cells (NSCs) generate neurons. In this study, we found that Notch3 plays an important role in the maintenance of quiescent NSCs (qNSCs), while Notch1 has been reported to act as a regulator

DEVELOPMENT OF AN INTACT HEPATOCYTE ACTIVATION SYSTEM FOR ROUTINE USE WITH THE MOUSE LYMPHOMA ASSAY

EPA Science Inventory

The authors have developed a method for cocultivating primary rat hepatocytes with L5178Y/TK+/- 3.7.2C mouse lymphoma cells. The system should provide a means to simulate more closely in vivo metabolism compared to metabolism by liver homogenates, while still being useful for rou...

Purification of Oogonial Stem Cells From Adult Mouse and Human Ovaries: An Assessment of the Literature and a View Toward the Future

PubMed Central

Woods, Dori C.; White, Yvonne A. R.; Tilly, Jonathan L.

2013-01-01

Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function. PMID:23024060

Purification of oogonial stem cells from adult mouse and human ovaries: an assessment of the literature and a view toward the future.

PubMed

Woods, Dori C; White, Yvonne A R; Tilly, Jonathan L

2013-01-01

Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function.

Immunostaining, dehydration, and clearing of mouse embryos for ultramicroscopy.

PubMed

Becker, Klaus; Jährling, Nina; Saghafi, Saiedeh; Dodt, Hans-Ulrich

2013-08-01

This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethanol and then cleared in a solution of benzyl alcohol and benzyl benzoate.

The IntAct molecular interaction database in 2012

PubMed Central

Kerrien, Samuel; Aranda, Bruno; Breuza, Lionel; Bridge, Alan; Broackes-Carter, Fiona; Chen, Carol; Duesbury, Margaret; Dumousseau, Marine; Feuermann, Marc; Hinz, Ursula; Jandrasits, Christine; Jimenez, Rafael C.; Khadake, Jyoti; Mahadevan, Usha; Masson, Patrick; Pedruzzi, Ivo; Pfeiffenberger, Eric; Porras, Pablo; Raghunath, Arathi; Roechert, Bernd; Orchard, Sandra; Hermjakob, Henning

2012-01-01

IntAct is an open-source, open data molecular interaction database populated by data either curated from the literature or from direct data depositions. Two levels of curation are now available within the database, with both IMEx-level annotation and less detailed MIMIx-compatible entries currently supported. As from September 2011, IntAct contains approximately 275 000 curated binary interaction evidences from over 5000 publications. The IntAct website has been improved to enhance the search process and in particular the graphical display of the results. New data download formats are also available, which will facilitate the inclusion of IntAct's data in the Semantic Web. IntAct is an active contributor to the IMEx consortium (http://www.imexconsortium.org). IntAct source code and data are freely available at http://www.ebi.ac.uk/intact. PMID:22121220

Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters.

PubMed

Contreras, Laura; Urbieta, Almudena; Kobayashi, Keiko; Saheki, Takeyori; Satrústegui, Jorgina

2010-04-01

The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons. (c) 2009 Wiley-Liss, Inc.

Role of Aquaporin-4 in Airspace-to-Capillary Water Permeability in Intact Mouse Lung Measured by a Novel Gravimetric Method

PubMed Central

Song, Yuanlin; Ma, Tonghui; Matthay, Michael A.; Verkman, A.S.

2000-01-01

a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport. PMID:10613915

Maternal choline supplementation programs greater activity of the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in adult Ts65Dn trisomic mice.

PubMed

Yan, Jian; Ginsberg, Stephen D; Powers, Brian; Alldred, Melissa J; Saltzman, Arthur; Strupp, Barbara J; Caudill, Marie A

2014-10-01

Maternal choline supplementation (MCS) induces lifelong cognitive benefits in the Ts65Dn mouse, a trisomic mouse model of Down syndrome and Alzheimer's disease. To gain insight into the mechanisms underlying these beneficial effects, we conducted a study to test the hypothesis that MCS alters choline metabolism in adult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic (2N) female littermates born to choline-unsupplemented or choline-supplemented Ts65Dn dams. Enrichment of d9-choline metabolites (derived from intact choline) and d3 + d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethanolamine N-methyltransferase (PEMT)] was measured in harvested tissues. Adult offspring (both Ts65Dn and 2N) of choline-supplemented (vs. choline-unsupplemented) dams exhibited 60% greater (P≤0.007) activity of hepatic PEMT, which functions in de novo choline synthesis and produces phosphatidylcholine (PC) enriched in docosahexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver, plasma, and brain in both genotypes but to a greater extent in the Ts65Dn adult offspring. MCS also yielded higher (P<0.05) d9 metabolite enrichments in liver, plasma, and brain. These data demonstrate that MCS exerts lasting effects on offspring choline metabolism, including up-regulation of the hepatic PEMT pathway and enhanced provision of choline and PEMT-PC to the brain. © FASEB.

50 CFR 622.381 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise be...

50 CFR 622.381 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise be...

50 CFR 622.276 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated, gilled...

50 CFR 622.276 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated, gilled...

Body condition and habitat use by Hermann's tortoises in burnt and intact habitats

PubMed Central

Lecq, S.; Ballouard, J.-M.; Caron, S.; Livoreil, B.; Seynaeve, V.; Matthieu, L.-A.; Bonnet, X.

2014-01-01

In Mediterranean regions, fires threaten terrestrial tortoises. Nevertheless, varying proportions of adults survive fire; these surviving individuals can play a central role for population recovery. The regions devastated by fire often include important habitat of Hermann's tortoises (Testudo hermanni hermanni), so assessing the ability of survivors to persist is essential for conserving the species. Body-condition indices provide an integrative estimate of how well individuals cope with environmental variations and impacts, including fires. Between 2002 and 2009, we monitored Hermann's tortoises in intact and burnt habitats in southeastern France. In summer 2003, a strong fire ravaged half of the surveyed zone, providing an opportunity to compare body condition of tortoises between intact and burnt areas over time. Six years later, the impact of fire on vegetation was still marked; large trees were abundant in the intact area, whereas open shrub vegetation prevailed in the burnt area. In both areas, the mean body condition of tortoises fluctuated over time; however, there were no differences between the two areas. A radio-tracking experiment demonstrated that individuals from each area were residents, and not vagrants commuting between areas. We also assessed changes in body condition and microhabitat use in radio-tracked individuals. We found no significant differences between the tortoises living in the burnt and intact areas, despite subtle differences in habitat use. In conclusion: (i) surviving tortoises in an area ravaged by fire can maintain their body condition like individuals living in an intact area, and thus, individuals from burnt areas should not be translocated to supposedly better areas; and (ii) depopulated burnt areas are likely to be appropriate for population-augmentation programmes. PMID:27293640

Sex differences in stress-induced social withdrawal: independence from adult gonadal hormones and inhibition of female phenotype by corncob bedding.

PubMed

Trainor, Brian C; Takahashi, Elizabeth Y; Campi, Katharine L; Florez, Stefani A; Greenberg, Gian D; Laman-Maharg, Abigail; Laredo, Sarah A; Orr, Veronica N; Silva, Andrea L; Steinman, Michael Q

2013-03-01

There is compelling evidence for important sex differences in behavioral and hormonal responses to psychosocial stress. Here we examined the effects of gonadal hormones on behavioral responses to social defeat stress in monogamous California mice (Peromyscus californicus). Three episodes of social defeat induced social withdrawal in intact females but not males. Gonadectomy blocked corticosterone responses to defeat in females and sensitized male corticosterone responses. However, gonadectomy had no effects on social interaction behavior, suggesting that social withdrawal is not dependent on gonadal hormones in the adult California mouse. In contrast, defeat reduced exploratory behavior in the open field test for intact but not castrated males. We also examined the effects of social defeat on social interaction behavior when California mice were raised on corncob bedding, which has estrogenic properties. In this dataset of over 300 mice, we observed that social defeat did not induce social withdrawal when females were raised on corncob bedding. This finding suggests that the use of corncob in rodent studies could mask important sex differences in the effects of stress on brain and behavior. Although gonadal hormones do not affect social withdrawal behavior in adults, our data suggest that hormones may act earlier in development to induce a more resilient social phenotype. Copyright © 2013 Elsevier Inc. All rights reserved.

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 50 Wildlife and Fisheries 12 2012-10-01 2012-10-01 false Landing fish intact. 622.38 Section 622... § 622.38 Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish on that vessel in the EEZ are maintained intact and, if taken from the EEZ, are...

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Landing fish intact. 622.38 Section 622... § 622.38 Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish on that vessel in the EEZ are maintained intact and, if taken from the EEZ, are...

Safety and efficacy of a ginkgo biloba-containing dietary supplement on cognitive function, quality of life, and platelet function in healthy, cognitively intact older adults.

PubMed

Carlson, Joseph J; Farquhar, John W; DiNucci, Ellen; Ausserer, Laurie; Zehnder, James; Miller, Donald; Berra, Kathy; Hagerty, Lisa; Haskell, William L

2007-03-01

To determine if a ginkgo biloba-containing supplement improves cognitive function and quality of life, alters primary hemostasis, and is safe in healthy, cognitively intact older adults. Four-month, randomized, double-blind, placebo-controlled parallel design. Ninety men and women (age range 65 to 84 years) were recruited to a university clinic. Eligibility included those without dementia or depression, not taking psychoactive medications or medications or supplements that alter hemostasis. Ninety subjects were randomly assigned to placebo or a ginkgo biloba-based supplement containing 160 mg ginkgo biloba, 68 mg gotu kola, and 180 mg decosahexaenoic acid per day for 4 months. Assessments included: six standardized cognitive function tests, the SF-36 Quality of Life questionnaire, the Platelet Function Analyzer-100 (Dade Behring, Eschbom, Germany), and the monitoring of adverse events. Baseline characteristics and study hypotheses were tested using analysis of covariance. Tests were two-tailed with a 0.05 significance level. Seventy-eight subjects (87%) completed both baseline and 4-month testing (n=36 in placebo group, n=42 in ginkgo biloba group). At baseline, the participants' cognitive function was above average. One of six cognitive tests indicated significant protocol differences at 4 months (P=0.03), favoring the placebo. There were no significant differences in quality of life, platelet function, or adverse events. These finding do not support the use of a ginkgo biloba-containing supplement for improving cognitive function or quality of life in cognitively intact, older, healthy adults. However, high baseline scores may have contributed to the null findings. The ginkgo biloba product seems safe and did not alter platelet function, though additional studies are needed to evaluate the interaction of varying doses of ginkgo biloba and ginkgo biloba-containing supplements with medications and supplements that alter hemostasis.

True navigation in migrating gulls requires intact olfactory nerves.

PubMed

Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A; Huttunen, Markku J; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

2015-11-24

During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances.

Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas

PubMed Central

Rovira, Meritxell; Scott, Sherri-Gae; Liss, Andrew S.; Jensen, Jan; Thayer, Sarah P.; Leach, Steven D.

2009-01-01

The question of whether dedicated progenitor cells exist in adult vertebrate pancreas remains controversial. Centroacinar cells and terminal duct (CA/TD) cells lie at the junction between peripheral acinar cells and the adjacent ductal epithelium, and are frequently included among cell types proposed as candidate pancreatic progenitors. However these cells have not previously been isolated in a manner that allows formal assessment of their progenitor capacities. We have found that a subset of adult CA/TD cells are characterized by high levels of ALDH1 enzymatic activity, related to high-level expression of both Aldh1a1 and Aldh1a7. This allows their isolation by FACS using a fluorogenic ALDH1 substrate. FACS-isolated CA/TD cells are relatively depleted of transcripts associated with differentiated pancreatic cell types. In contrast, they are markedly enriched for transcripts encoding Sca1, Sdf1, c-Met, Nestin, and Sox9, markers previously associated with progenitor populations in embryonic pancreas and other tissues. FACS-sorted CA/TD cells are uniquely able to form self-renewing “pancreatospheres” in suspension culture, even when plated at clonal density. These spheres display a capacity for spontaneous endocrine and exocrine differentiation, as well as glucose-responsive insulin secretion. In addition, when injected into cultured embryonic dorsal pancreatic buds, these adult cells display a unique capacity to contribute to both the embryonic endocrine and exocrine lineages. Finally, these cells demonstrate dramatic expansion in the setting of chronic epithelial injury. These findings suggest that CA/TD cells are indeed capable of progenitor function and may contribute to the maintenance of tissue homeostasis in adult mouse pancreas. PMID:20018761

Adult Plasticity in the Subcortical Auditory Pathway of the Maternal Mouse

PubMed Central

Miranda, Jason A.; Shepard, Kathryn N.; McClintock, Shannon K.; Liu, Robert C.

2014-01-01

Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system – motherhood – is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered. PMID:24992362

Adult plasticity in the subcortical auditory pathway of the maternal mouse.

PubMed

Miranda, Jason A; Shepard, Kathryn N; McClintock, Shannon K; Liu, Robert C

2014-01-01

Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered.

Ultrasonic vocalizations of adult male Foxp2-mutant mice: behavioral contexts of arousal and emotion.

PubMed

Gaub, S; Fisher, S E; Ehret, G

2016-02-01

Adult mouse ultrasonic vocalizations (USVs) occur in multiple behavioral and stimulus contexts associated with various levels of arousal, emotion and social interaction. Here, in three experiments of increasing stimulus intensity (water; female urine; male interacting with adult female), we tested the hypothesis that USVs of adult males express the strength of arousal and emotion via different USV parameters (18 parameters analyzed). Furthermore, we analyzed two mouse lines with heterozygous Foxp2 mutations (R552H missense, S321X nonsense), known to produce severe speech and language disorders in humans. These experiments allowed us to test whether intact Foxp2 function is necessary for developing full adult USV repertoires, and whether mutations of this gene influence instinctive vocal expressions based on arousal and emotion. The results suggest that USV calling rate characterizes the arousal level, while sound pressure and spectrotemporal call complexity (overtones/harmonics, type of frequency jumps) may provide indices of levels of positive emotion. The presence of Foxp2 mutations did not qualitatively affect the USVs; all USV types that were found in wild-type animals also occurred in heterozygous mutants. However, mice with Foxp2 mutations displayed quantitative differences in USVs as compared to wild-types, and these changes were context dependent. Compared to wild-type animals, heterozygous mutants emitted mainly longer and louder USVs at higher minimum frequencies with a higher occurrence rate of overtones/harmonics and complex frequency jump types. We discuss possible hypotheses about Foxp2 influence on emotional vocal expressions, which can be investigated in future experiments using selective knockdown of Foxp2 in specific brain circuits. © 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Cardiac muscle organization revealed in 3-D by imaging whole-mount mouse hearts using two-photon fluorescence and confocal microscopy.

PubMed

Sivaguru, Mayandi; Fried, Glenn; Sivaguru, Barghav S; Sivaguru, Vignesh A; Lu, Xiaochen; Choi, Kyung Hwa; Saif, M Taher A; Lin, Brian; Sadayappan, Sakthivel

2015-11-01

The ability to image the entire adult mouse heart at high resolution in 3-D would provide enormous advantages in the study of heart disease. However, a technique for imaging nuclear/cellular detail as well as the overall structure of the entire heart in 3-D with minimal effort is lacking. To solve this problem, we modified the benzyl alcohol:benzyl benzoate (BABB) clearing technique by labeling mouse hearts with periodic acid Schiff (PAS) stain. We then imaged the hearts with a combination of two-photon fluorescence microscopy and automated tile-scan imaging/stitching. Utilizing the differential spectral properties of PAS, we could identify muscle and nuclear compartments in the heart. We were also able to visualize the differences between a 3-month-old normal mouse heart and a mouse heart that had undergone heart failure due to the expression of cardiac myosin binding protein-C (cMyBP-C) gene mutation (t/t). Using 2-D and 3-D morphometric analysis, we found that the t/t heart had anomalous ventricular shape, volume, and wall thickness, as well as a disrupted sarcomere pattern. We further validated our approach using decellularized hearts that had been cultured with 3T3 fibroblasts, which were tracked using a nuclear label. We were able to detect the 3T3 cells inside the decellularized intact heart tissue, achieving nuclear/cellular resolution in 3-D. The combination of labeling, clearing, and two-photon microscopy together with tiling eliminates laborious and time-consuming physical sectioning, alignment, and 3-D reconstruction.

Proteolysis breaks tolerance toward intact α345(IV) collagen, eliciting novel anti-GBM autoantibodies specific for α345NC1 hexamers

PubMed Central

Olaru, Florina; Wang, Xu-Ping; Luo, Wentian; Ge, Linna; Miner, Jeffrey H; Kleinau, Sandra; Geiger, Xochiquetzal J.; Wasiluk, Andrew; Heidet, Laurence; Kitching, A. Richard; Borza, Dorin-Bogdan

2012-01-01

Goodpasture disease is an autoimmune kidney disease mediated by autoAbs against NC1 monomers of α3(IV) collagen that bind to the glomerular basement membrane (GBM), usually causing rapidly progressive glomerulonephritis. We identified a novel type of human IgG4-restricted anti-GBM autoAbs associated with mild non-progressive glomerulonephritis, which specifically targeted α345NC1 hexamers but not α3NC1 monomers. The mechanisms eliciting these anti-GBM autoAbs were investigated in mouse models recapitulating this phenotype. Wild type and FcγRIIB−/− mice immunized with autologous murine GBM NC1 hexamers produced mouse IgG1-restricted autoAbs specific for α345NC1 hexamers, which bound to the GBM in vivo but did not cause glomerulonephritis. In these mice, intact collagen IV from murine GBM was not immunogenic. However, in Col4a3−/− Alport mice, both intact collagen IV and NC1 hexamers from murine GBM elicited IgG antibodies specific for α3α4α5NC1 hexamers, which were not subclass restricted. As heterologous antigen in COL4A3-humanized mice, murine GBM NC1 hexamers elicited mouse IgG1, IgG2a and IgG2b autoAbs specific for α345NC1 hexamers and induced anti-GBM Ab glomerulonephritis. These findings indicate that tolerance toward autologous intact α3α4α5(IV) collagen is established in hosts expressing this antigen, even though autoreactive B cells specific for α345NC1 hexamers are not purged from their repertoire. Proteolysis selectively breaches this tolerance by generating autoimmunogenic α3α4α5NC1 hexamers. This provides a mechanism eliciting autoAbs specific for α345NC1 hexamers, which are restricted to non-inflammatory IgG subclasses and non-nephritogenic. In Alport syndrome, lack of tolerance toward α3α4α5(IV) collagen promotes production of alloantibodies to α345NC1 hexamers, including pro-inflammatory IgG subclasses which mediate post-transplant anti-GBM nephritis. PMID:23303673

Development of Second Generation Intact Stability Criteria

DTIC Science & Technology

2011-12-01

the intact stability performance of ships, have motivated the development of the second generation intact stability criteria by the IMO Subcommittee on...primary modes of stability failures which are being addressed. The second generation intact stability criteria are planned to have a multitiered structure...and 2 vulnerability criteria that are used as a preliminary design process check of dynamic stability failure risk. This report describes the U.S

Presence of orally administered rice bran oil γ-oryzanol in its intact form in mouse plasma.

PubMed

Kobayashi, Eri; Ito, Junya; Kato, Shunji; Sawada, Kazue; Matsuki, Midori; Hashimoto, Hiroyuki; Miyazawa, Teruo; Nakagawa, Kiyotaka

2016-12-07

Although the beneficial effects (e.g., lipid-lowering activity) of γ-oryzanol (OZ), a mixture of ferulic acid esters of plant sterols and triterpene alcohols, have been extensively investigated, few studies have evaluated the absorption and metabolism of OZ. Moreover, it is unclear whether OZ, once ingested, is directly absorbed by the intestine into the bloodstream at a sufficient level to exhibit activity. Here, we prepared OZ concentrate from purified rice bran oil (Rice Oil OZ), determined the concentration of OZ in the preparation (cycloartenyl ferulate equivalent concentration; 52.2%), and then carried out chromatography-mass spectrometry analysis of plasma samples from mice after oral administration of Rice Oil OZ. The OZ concentrations of plasma from the control (vehicle-treated) mice were low (trace levels); however, at 5 h after a single oral administration of the Rice Oil OZ (600 mg per kg body weight), the levels significantly increased, reaching 17.6 ng mL -1 for cycloartenyl ferulate, 28.2 ng mL -1 for 24-methylenecycloartanyl ferulate isomers, 15.6 ng mL -1 for campesteryl ferulate, and 5.1 ng mL -1 for β-sitosteryl ferulate, respectively, expressed in equivalence of cycloartenyl ferulate in plasma. These results provided the first mass spectrometric evidence suggesting that a portion of orally administered OZ is directly absorbed by the intestine and is present in the intact form in plasma. The presence of a significant amount of OZ in its intact form in plasma may explain the beneficial effects of OZ in vivo.

Intact parathyroid hormone and whole parathyroid hormone assay results disagree in hemodialysis patients under cinacalcet hydrochloride therapy.

PubMed

Koda, Ryo; Kazama, Junichiro James; Matsuo, Koji; Kawamura, Kazuko; Yamamoto, Suguru; Wakasugi, Minako; Takeda, Tetsuro; Narita, Ichiei

2015-08-01

The parathyroid gland secretes 1-84 and 7-84 parathyroid hormone (PTH) fragments, and its regulation is dependent on stimulation of the extracellular calcium-sensing receptor. While the intact PTH system detects both PTH fragments, the whole PTH system detects the 1-84PTH but not the 7-84PTH. Cinacalcet hydrochloride (CH) binds to calcium-sensing receptor as a calcimimetic. Here we investigated the role of CH treatment in the assessment of parathyroid gland function. Stable adult dialysis patients for whom CH therapy was planned were included. Patients for whom CH therapy was not planned were simultaneously included as the control group. The CH group (n = 44) showed significantly higher circulating levels of Ca, intact PTH, and whole PTH, before the CH treatment than the control group (n = 112). The Ca, intact PTH, and whole PTH levels decreased along with the CH therapy, and the Ca levels became comparable in the 8th week of treatment and thereafter. The CH group in the 8th week and thereafter showed significantly lower whole/intact PTH ratios than the control group, while the whole/intact PTH ratio was not significantly different between before and during the CH therapy. A multiple regression analysis revealed that the whole/intact PTH ratio was almost constant, but both the serum Ca level and a CH therapy could potentially modify the fixed number. When the whole PTH levels were estimated by intact PTH levels using the relationship between them in the control group, the levels were clearly overestimated in the CH group. Although the direct effect of CH on the whole/intact PTH ratio is masked by its hypocalcemic action, we could successfully demonstrate that the ratio in CH users is lower than that in the non-users with comparable levels of serum Ca. Evaluating parathyroid function with intact PTH according to the clinical practice guidelines in patients being treated with CH may lead to significant overestimation and subsequent overtreatment.

Intraindividual variability in cognitive performance in older adults: comparison of adults with mild dementia, adults with arthritis, and healthy adults.

PubMed

Hultsch, D F; MacDonald, S W; Hunter, M A; Levy-Bencheton, J; Strauss, E

2000-10-01

Intraindividual variability in latency and accuracy of cognitive performance across both trials and occasions was examined in 3 groups of older adults: healthy adults, adults with arthritis, and adults diagnosed with mild dementia. Participants completed 2 reaction-time and 2 episodic-memory tasks on 4 occasions. Results indicated that intraindividual variability in latency was greater in individuals diagnosed with mild dementia than in adults who were neurologically intact, regardless of their health status. Individual differences in variability were stable over time and across cognitive domains. Intraindividual variability was also related to level of performance and was uniquely predictive of neurological status, independent of level of performance. Results suggest that intraindividual variability may be a behavioral indicator of compromised neurological mechanisms.

RUNX1B Expression Is Highly Heterogeneous and Distinguishes Megakaryocytic and Erythroid Lineage Fate in Adult Mouse Hematopoiesis

PubMed Central

Draper, Julia E.; Sroczynska, Patrycja; Tsoulaki, Olga; Leong, Hui Sun; Fadlullah, Muhammad Z. H.; Miller, Crispin; Kouskoff, Valerie; Lacaud, Georges

2016-01-01

The Core Binding Factor (CBF) protein RUNX1 is a master regulator of definitive hematopoiesis, crucial for hematopoietic stem cell (HSC) emergence during ontogeny. RUNX1 also plays vital roles in adult mice, in regulating the correct specification of numerous blood lineages. Akin to the other mammalian Runx genes, Runx1 has two promoters P1 (distal) and P2 (proximal) which generate distinct protein isoforms. The activities and specific relevance of these two promoters in adult hematopoiesis remain to be fully elucidated. Utilizing a dual reporter mouse model we demonstrate that the distal P1 promoter is broadly active in adult hematopoietic stem and progenitor cell (HSPC) populations. By contrast the activity of the proximal P2 promoter is more restricted and its upregulation, in both the immature Lineage- Sca1high cKithigh (LSK) and bipotential Pre-Megakaryocytic/Erythroid Progenitor (PreMegE) populations, coincides with a loss of erythroid (Ery) specification. Accordingly the PreMegE population can be prospectively separated into “pro-erythroid” and “pro-megakaryocyte” populations based on Runx1 P2 activity. Comparative gene expression analyses between Runx1 P2+ and P2- populations indicated that levels of CD34 expression could substitute for P2 activity to distinguish these two cell populations in wild type (WT) bone marrow (BM). Prospective isolation of these two populations will enable the further investigation of molecular mechanisms involved in megakaryocytic/erythroid (Mk/Ery) cell fate decisions. Having characterized the extensive activity of P1, we utilized a P1-GFP homozygous mouse model to analyze the impact of the complete absence of Runx1 P1 expression in adult mice and observed strong defects in the T cell lineage. Finally, we investigated how the leukemic fusion protein AML1-ETO9a might influence Runx1 promoter usage. Short-term AML1-ETO9a induction in BM resulted in preferential P2 upregulation, suggesting its expression may be important to

Rhythmic ganglion cell activity in bleached and blind adult mouse retinas.

PubMed

Menzler, Jacob; Channappa, Lakshmi; Zeck, Guenther

2014-01-01

In retinitis pigmentosa--a degenerative disease which often leads to incurable blindness--the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor's dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the

Equol inhibits growth, induces atresia, and inhibits steroidogenesis of mouse antral follicles in vitro

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

Mahalingam, Sharada, E-mail: mahalin2@illinois.edu; Gao, Liying, E-mail: lgao@uiuc.edu; Gonnering, Marni, E-mail: mgonne2@illinois.edu

Equol is a non-steroidal estrogen metabolite produced by microbial conversion of daidzein, a major soy isoflavone, in the gut of some humans and many animal species. Isoflavones and their metabolites can affect endogenous estradiol production, action, and metabolism, potentially influencing ovarian follicle function. However, no studies have examined the effects of equol on intact ovarian antral follicles, which are responsible for sex steroid synthesis and further development into ovulatory follicles. Thus, the present study tested the hypothesis that equol inhibits antral follicle growth, increases follicle atresia, and inhibits steroidogenesis in the adult mouse ovary. To test this hypothesis, antral folliclesmore » isolated from adult CD-1 mice were cultured with vehicle control (dimethyl sulfoxide; DMSO) or equol (600 nM, 6 μM, 36 μM, and 100 μM) for 48 and 96 h. Every 24 h, follicle diameters were measured to monitor growth. At 48 and 96 h, the culture medium was subjected to measurement of hormone levels, and the cultured follicles were subjected to gene expression analysis. Additionally, follicles were histologically evaluated for signs of atresia after 96 h of culture. The results indicate that equol (100 μM) inhibited follicle growth, altered the mRNA levels of bcl2-associated X protein and B cell leukemia/lymphoma 2, and induced follicle atresia. Further, equol decreased the levels of estradiol, testosterone, androstenedione, and progesterone, and it decreased mRNA levels of cholesterol side-chain cleavage, steroid 17-α-hydroxalase, and aromatase. Collectively, these data indicate that equol inhibits growth, increases atresia, and inhibits steroidogenesis of cultured mouse antral follicles. - Highlights: • Equol exposure inhibits antral follicle growth. • Equol exposure increases follicle atresia. • Equol exposure inhibits sex steroid hormone levels. • Equol exposure inhibits mRNA levels of certain steroidogenic enzymes.« less

Irradiation at Different Fetal Stages Results in Different Translocation Frequencies in Adult Mouse Thyroid Cells

DOE PAGES

Hamasaki, K.; Landes, R. D.; Noda, A.; ...

2016-10-01

While it is generally believed that fetuses are at high risk of developing cancers, including leukemia, after low doses of radiation, it has been reported that atomic bomb survivors exposed in utero did not show a dose response for translocations in blood T lymphocytes when they were examined at approximately 40 years of age. Subsequent mouse studies confirmed that animals irradiated during the fetal stage did not show evidence of radiation effects in lymphocytes and bone marrow cells when they were examined after reaching adulthood. However, in a study of rat mammary epithelial cells, radiation effects were clearly observed aftermore » fetal irradiation. These results indicate that the fate of chromosome aberrations induced in a fetus could vary among different tissues. Here we report on translocation frequencies in mouse thyroid cells, which were irradiated at different stages of fetal development. Cytogenetic examination was then conducted using fluorescence in situ hybridization (FISH) painting of chromosomes 1 and 3. Adult mice, 2 Gy X-ray irradiated at 15.5-day-old fetuses (E15.5), showed a higher translocation frequency (30/1,155 or 25.3 x 10 -3) than nonirradiated adult controls (0/1,007 or 0.1 x 10 -3), and was near that experienced by irradiated mothers and non-pregnant adult females (43/1,244 or 33.7 x 10 -3). These results are consistent with those seen in rat mammary cells. However, when fetuses were irradiated at an earlier stage of development (E6.5) before thyroid organogenesis, the resulting observed translocation frequency was much lower (3/502 or 5.8 x 10 -3) than that in E15.5 mice. These results suggest that after fetal irradiation, tissue stem cells record radiation effects primarily when the exposure occurs in cells that have been integrated into tissue. Embryonic stem cells that have been damaged prior to integration into the niche may undergo negative selection due to apoptosis, mitotic death or stem cell-niche cell interactions. The

Long-term administration of scopolamine interferes with nerve cell proliferation, differentiation and migration in adult mouse hippocampal dentate gyrus, but it does not induce cell death

PubMed Central

Yan, Bing Chun; Park, Joon Ha; Chen, Bai Hui; Cho, Jeong-Hwi; Kim, In Hye; Ahn, Ji Hyeon; Lee, Jae-Chul; Hwang, In Koo; Cho, Jun Hwi; Lee, Yun Lyul; Kang, Il-Jun; Won, Moo-Ho

2014-01-01

Long-term administration of scopolamine, a muscarinic receptor antagonist, can inhibit the survival of newly generated cells, but its effect on the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus remain poorly understood. In this study, we used immunohistochemistry and western blot methods to weekly detect the biological behaviors of nerve cells in the hippocampal dentate gyrus of adult mice that received intraperitoneal administration of scopolamine for 4 weeks. Expression of neuronal nuclear antigen (NeuN; a neuronal marker) and Fluoro-Jade B (a marker for the localization of neuronal degeneration) was also detected. After scopolamine treatment, mouse hippocampal neurons did not die, and Ki-67 (a marker for proliferating cells)-immunoreactive cells were reduced in number and reached the lowest level at 4 weeks. Doublecortin (DCX; a marker for newly generated neurons)-immunoreactive cells were gradually shortened in length and reduced in number with time. After scopolamine treatment for 4 weeks, nearly all of the 5-bromo-2′-deoxyuridine (BrdU)-labeled newly generated cells were located in the subgranular zone of the dentate gyrus, but they did not migrate into the granule cell layer. Few mature BrdU/NeuN double-labeled cells were seen in the subgranular zone of the dentate gyrus. These findings suggest that long-term administration of scopolamine interferes with the proliferation, differentiation and migration of nerve cells in the adult mouse hippocampal dentate gyrus, but it does not induce cell death. PMID:25422633

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

PubMed

Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

2005-08-01

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

True navigation in migrating gulls requires intact olfactory nerves

PubMed Central

Wikelski, Martin; Arriero, Elena; Gagliardo, Anna; Holland, Richard A.; Huttunen, Markku J.; Juvaste, Risto; Mueller, Inge; Tertitski, Grigori; Thorup, Kasper; Wild, Martin; Alanko, Markku; Bairlein, Franz; Cherenkov, Alexander; Cameron, Alison; Flatz, Reinhard; Hannila, Juhani; Hüppop, Ommo; Kangasniemi, Markku; Kranstauber, Bart; Penttinen, Maija-Liisa; Safi, Kamran; Semashko, Vladimir; Schmid, Heidi; Wistbacka, Ralf

2015-01-01

During migratory journeys, birds may become displaced from their normal migratory route. Experimental evidence has shown that adult birds can correct for such displacements and return to their goal. However, the nature of the cues used by migratory birds to perform long distance navigation is still debated. In this experiment we subjected adult lesser black-backed gulls migrating from their Finnish/Russian breeding grounds (from >60°N) to Africa (to < 5°N) to sensory manipulation, to determine the sensory systems required for navigation. We translocated birds westward (1080 km) or eastward (885 km) to simulate natural navigational challenges. When translocated westwards and outside their migratory corridor birds with olfactory nerve section kept a clear directional preference (southerly) but were unable to compensate for the displacement, while intact birds and gulls with the ophthalmic branch of the trigeminal nerve sectioned oriented towards their population-specific migratory corridor. Thus, air-borne olfactory information seems to be important for migrating gulls to navigate successfully in some circumstances. PMID:26597351

Quantifying growing versus non-growing ovarian follicles in the mouse.

PubMed

Uslu, Bahar; Dioguardi, Carola Conca; Haynes, Monique; Miao, De-Qiang; Kurus, Meltem; Hoffman, Gloria; Johnson, Joshua

2017-01-13

A standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these 'historical' criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying). Reasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries. We found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation. Combined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

In vivo robotics: the automation of neuroscience and other intact-system biological fields.

PubMed

Kodandaramaiah, Suhasa B; Boyden, Edward S; Forest, Craig R

2013-12-01

Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to influence neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience and present a concrete example with our recent automation of in vivo whole-cell patch clamp electrophysiology of neurons in the living mouse brain. © 2013 New York Academy of Sciences.

In vivo robotics: the automation of neuroscience and other intact-system biological fields

PubMed Central

Kodandaramaiah, Suhasa B.; Boyden, Edward S.; Forest, Craig R.

2013-01-01

Robotic and automation technologies have played a huge role in in vitro biological science, having proved critical for scientific endeavors such as genome sequencing and high-throughput screening. Robotic and automation strategies are beginning to play a greater role in in vivo and in situ sciences, especially when it comes to the difficult in vivo experiments required for understanding the neural mechanisms of behavior and disease. In this perspective, we discuss the prospects for robotics and automation to impact neuroscientific and intact-system biology fields. We discuss how robotic innovations might be created to open up new frontiers in basic and applied neuroscience, and present a concrete example with our recent automation of in vivo whole cell patch clamp electrophysiology of neurons in the living mouse brain. PMID:23841584

Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders

PubMed Central

Scattoni, Maria Luisa; Crawley, Jacqueline; Ricceri, Laura

2009-01-01

In neonatal mice ultrasonic vocalizations have been studied both as an early communicative behavior of the pup-mother dyad and as a sign of an aversive affective state. Adult mice of both sexes produce complex ultrasonic vocalization patterns in different experimental/social contexts. All these vocalizations are becoming an increasingly valuable assay for behavioral phenotyping throughout the mouse life-span and alterations of the ultrasound patterns have been reported in several mouse models of neurodevelopmental disorders. Here we also show that the modulation of vocalizations by maternal cues (maternal potentiation paradigm) – originally identified and investigated in rats - can be measured in C57Bl/6 mouse pups with appropriate modifications of the rat protocol and can likely be applied to mouse behavioral phenotyping. In addition we suggest that a detailed qualitative evaluation of neonatal calls together with analysis of adult mouse vocalization patterns in both sexes in social settings, may lead to a greater understanding of the communication value of vocalizations in mice. Importantly, both neonatal and adult USV altered patterns can be determined during the behavioural phenotyping of mouse models of human neurodevelopmental and neuropsychiatric disorders, starting from those in which deficits in communication are a primary symptom. PMID:18771687

Monoacylglycerol lipase inhibitor JZL184 reduces neuroinflammatory response in APdE9 mice and in adult mouse glial cells.

PubMed

Pihlaja, Rea; Takkinen, Jatta; Eskola, Olli; Vasara, Jenni; López-Picón, Francisco R; Haaparanta-Solin, Merja; Rinne, Juha O

2015-04-28

Recently, the role of monoacylglycerol lipase (MAGL) as the principal regulator of simultaneous prostaglandin synthesis and endocannabinoid receptor activation in the CNS was demonstrated. To expand upon previously published research in the field, we observed the effect of the MAGL inhibitor JZL184 during the early-stage proinflammatory response and formation of beta-amyloid (Aβ) in the Alzheimer's disease mouse model APdE9. We also investigated its effects in proinflammatory agent - induced astrocytes and microglia isolated from adult mice. Transgenic APdE9 mice (5 months old) were treated with JZL184 (40 mg/kg) or vehicle every day for 1 month. In vivo binding of the neuroinflammation-related, microglia-specific translocator protein (TSPO) targeting radioligand [(18) F]GE-180 decreased slightly but statistically non-significantly in multiple brain areas compared to vehicle-treated mice. JZL184 treatment induced a significant decrease in expression levels of inflammation-induced, Iba1-immunoreactive microglia in the hippocampus (P < 0.01) and temporal and parietal (P < 0.05) cortices. JZL184 also induced a marked decrease in total Aβ burden in the temporal (P < 0.001) and parietal (P < 0.01) cortices and, to some extent, in the hippocampus. Adult microglial and astrocyte cultures pre-treated with JZL184 and then exposed to the neuroinflammation-inducing agents lipopolysaccharide (LPS), interferon-gamma (IFN-γ), and Aβ42 had significantly reduced proinflammatory responses compared to cells without JZL184 treatment. JZL184 decreased the proinflammatory reactions of microglia and reduced the total Aβ burden and its precursors in the APdE9 mouse model. It also reduced the proinflammatory responses of microglia and astrocytes isolated from adult mice.

A randomized trial to measure the impact of a community-based cognitive training intervention on balance and gait in cognitively intact Black older adults.

PubMed

Smith-Ray, Renae L; Makowski-Woidan, Beth; Hughes, Susan L

2014-10-01

Fall prevention is important for maintaining mobility and independence into old age. Approaches for reducing falls include exercise, tai chi, and home modifications; however, causes of falling are multifactorial and include not just physical but cognitive factors. Cognitive decline occurs with age, but older adults with the greatest declines in executive function experience more falls. The purpose of this study was twofold: to demonstrate the feasibility of a community-based cognitive training program for cognitively intact Black older adults and to analyze its impact on gait and balance in this population. This pilot study used a pretest/posttest randomized trial design with assignment to an intervention or control group. Participants assigned to the intervention completed a computer-based cognitive training class that met 2 days a week for 60 min over 10 weeks. Classes were held at senior/community centers. Primary outcomes included balance as measured by the Berg Balance Scale (BBS), 10-meter gait speed, and 10-meter gait speed under visuospatial dual-task condition. All measures were assessed at baseline and immediately post-intervention. Participants were community-dwelling Black adults with a mean age of 72.5 and history of falls (N = 45). Compared to controls, intervention participants experienced statistically significant improvements in BBS and gait speed. Mean performance on distracted gait speed also improved more for intervention participants compared to controls. Findings from this pilot randomized trial demonstrate the feasibility of a community-based cognitive training intervention. They provide initial evidence that cognitive training may be an efficacious approach toward improving balance and gait in older adults known to have a history of falls. © 2014 Society for Public Health Education.

Mouse Models of Human T Lymphotropic Virus Type-1–Associated Adult T-Cell Leukemia/Lymphoma

PubMed Central

Zimmerman, B.; Niewiesk, S.; Lairmore, M. D.

2011-01-01

Human T-lymphotropic virus type-1 (HTLV-1), the first human retrovirus discovered, is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic. Mechanisms of early infection and cell-to-cell transmission can be studied in rabbits and nonhuman primates, but lesion development and reagents are limited in these species. The mouse provides a cost-effective, highly reproducible model in which to study factors related to lymphoma development and the preclinical efficacy of potential therapies against ATL. The ability to manipulate transgenic mice has provided important insight into viral genes responsible for lymphocyte transformation. Expansion of various strains of immunodeficient mice has accelerated the testing of drugs and targeted therapy against ATL. This review compares various mouse models to illustrate recent advances in the understanding of HTLV-1–associated ATL development and how improvements in these models are critical to the future development of targeted therapies against this aggressive T-cell lymphoma. PMID:20442421

Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse.

PubMed

Nordenankar, Karin; Bergfors, Assar; Wallén-Mackenzie, Åsa

2015-01-01

Anxiety is a natural emotion experienced by all individuals. However, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. Anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies. We previously demonstrated that mice lacking forebrain expression of the Vesicular glutamate transporter 2 (Vglut2) from adolescence showed a strong anxiolytic behaviour as adults. In the current study, we wished to analyse if removal of Vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse. We produced transgenic mice lacking Vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood. The transgenic mice lacking Vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when Vglut2 was removed during adolescence. Our results suggest that both embryonal and adolescent forebrain expression of Vglut2 normally contributes to balancing the level of anxiety. As the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans.

Taste Bud Labeling in Whole Tongue Epithelial Sheet in Adult Mice.

PubMed

Venkatesan, Nandakumar; Boggs, Kristin; Liu, Hong-Xiang

2016-04-01

Molecular labeling in whole-mount tissues provides an efficient way to obtain general information about the formation, maintenance, degeneration, and regeneration of many organs and tissues. However, labeling of lingual taste buds in whole tongue tissues in adult mice has been problematic because of the strong permeability barrier of the tongue epithelium. In this study, we present a simple method for labeling taste buds in the intact tongue epithelial sheet of an adult mouse. Following intralingual protease injection and incubation, immediate fixation of the tongue on mandible in 4% paraformaldehyde enabled the in situ shape of the tongue epithelium to be well maintained after peeling. The peeled epithelium was accessible to taste bud labeling with a pan-taste cell marker, keratin 8, and a type II taste cell marker, α-gustducin, in all three types of taste papillae, that is, fungiform, foliate, and circumvallate. Overnight incubation of tongue epithelial sheets with primary and secondary antibodies was sufficient for intense labeling of taste buds with both fluorescent and DAB visualizations. Labeled individual taste buds were easy to identify and quantify. This protocol provides an efficient way for phenotypic analyses of taste buds, especially regarding distribution pattern and number.

Head Transplantation in Mouse Model.

PubMed

Ren, Xiao-Ping; Ye, Yi-Jie; Li, Peng-Wei; Shen, Zi-Long; Han, Ke-Cheng; Song, Yang

2015-08-01

The mouse model of allo-head and body reconstruction (AHBR) has recently been established to further the clinical development of this strategy for patients who are suffering from mortal bodily trauma or disease, yet whose mind remains healthy. Animal model studies are indispensable for developing such novel surgical practices. The goal of this work was to establish head transplant mouse model, then the next step through the feasible biological model to investigate immune rejection and brain function in next step, thereby promoting the goal of translation of AHBR to the clinic in the future. Our approach involves retaining adequate blood perfusion in the transplanted head throughout the surgical procedure by establishing donor-to-recipient cross-circulation by cannulating and anastomosing the carotid artery on one side of the body and the jugular vein on the other side. Neurological function was preserved by this strategy as indicated by electroencephalogram and intact cranial nerve reflexes. The results of this study support the feasibility of this method for avoiding brain ischemia during transplantation, thereby allowing for the possibility of long-term studies of head transplantation. © 2015 John Wiley & Sons Ltd.

Thalamocortical Projection Neuron and Interneuron Numbers in the Visual Thalamic Nuclei of the Adult C57BL/6 Mouse.

PubMed

Evangelio, Marian; García-Amado, María; Clascá, Francisco

2018-01-01

A key parameter to constrain predictive, bottom-up circuit models of a given brain domain is the number and position of the neuronal populations involved. These include not only the neurons whose bodies reside within the domain, but also the neurons in distant regions that innervate the domain. The mouse visual cortex receives its main subcortical input from the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior (LP) complex of the thalamus. The latter consists of three different nuclei: lateral posterior lateral (LPL), lateral posterior medial rostral (LPMR), and lateral posterior medial caudal (LPMC), each exhibiting specific patterns of connections with the various visual cortical areas. Here, we have determined the number of thalamocortical projection neurons and interneurons in the LP complex and dLGN of the adult C57BL/6 male mouse. We combined Nissl staining and histochemical and immunolabeling methods for consistently delineating nuclei borders, and applied unbiased stereological cell counting methods. Thalamic interneurons were identified using GABA immunolabeling. The C57BL/6 dLGN contains ∼21,200 neurons, while LP complex contains ∼31,000 total neurons. The dLGN and LP are the only nuclei of the mouse dorsal thalamus containing substantial numbers GABA-immunoreactive interneurons. These interneurons, however, are scarcer than previously estimated; they are 5.6% of dLGN neurons and just 1.9% of the LP neurons. It can be thus inferred that the dLGN contains ∼20,000 and the LP complex ∼30,400 thalamocortical projection neurons (∼12,000 in LPL, 15,200 in LPMR, and 4,200 in LPMC). The present dataset is relevant for constraining models of mouse visual thalamocortical circuits, as well as for quantitative comparisons between genetically modified mouse strains, or across species.

Thalamocortical Projection Neuron and Interneuron Numbers in the Visual Thalamic Nuclei of the Adult C57BL/6 Mouse

PubMed Central

Evangelio, Marian; García-Amado, María; Clascá, Francisco

2018-01-01

A key parameter to constrain predictive, bottom-up circuit models of a given brain domain is the number and position of the neuronal populations involved. These include not only the neurons whose bodies reside within the domain, but also the neurons in distant regions that innervate the domain. The mouse visual cortex receives its main subcortical input from the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior (LP) complex of the thalamus. The latter consists of three different nuclei: lateral posterior lateral (LPL), lateral posterior medial rostral (LPMR), and lateral posterior medial caudal (LPMC), each exhibiting specific patterns of connections with the various visual cortical areas. Here, we have determined the number of thalamocortical projection neurons and interneurons in the LP complex and dLGN of the adult C57BL/6 male mouse. We combined Nissl staining and histochemical and immunolabeling methods for consistently delineating nuclei borders, and applied unbiased stereological cell counting methods. Thalamic interneurons were identified using GABA immunolabeling. The C57BL/6 dLGN contains ∼21,200 neurons, while LP complex contains ∼31,000 total neurons. The dLGN and LP are the only nuclei of the mouse dorsal thalamus containing substantial numbers GABA-immunoreactive interneurons. These interneurons, however, are scarcer than previously estimated; they are 5.6% of dLGN neurons and just 1.9% of the LP neurons. It can be thus inferred that the dLGN contains ∼20,000 and the LP complex ∼30,400 thalamocortical projection neurons (∼12,000 in LPL, 15,200 in LPMR, and 4,200 in LPMC). The present dataset is relevant for constraining models of mouse visual thalamocortical circuits, as well as for quantitative comparisons between genetically modified mouse strains, or across species. PMID:29706872

Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle.

PubMed

Raskin, Anna M; Hoshijima, Masahiko; Swanson, Eric; McCulloch, Andrew D; Omens, Jeffrey H

2009-09-01

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas.

PubMed

Valenti, Daniela; de Bari, Lidia; De Filippis, Bianca; Ricceri, Laura; Vacca, Rosa Anna

2014-01-01

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses. Copyright © 2013 Elsevier Inc. All rights reserved.

Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

PubMed Central

Tjondrokoesoemo, Andoria; Park, Ki Ho; Ferrante, Christopher; Komazaki, Shinji; Lesniak, Sebastian; Brotto, Marco; Ko, Jae-Kyun; Zhou, Jingsong; Weisleder, Noah; Ma, Jianjie

2011-01-01

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle

Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

PubMed Central

Hilgenberg, Jill D.; Rybalchenko, Volodymyr; Medina-Ortiz, Wanda E.; Gregg, Elaine V.; Koulen, Peter

2011-01-01

Purpose. Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca2+ homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca2+ concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca2+ release channels, transient receptor potential polycystin family (TRPP) channels. Methods. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca2+ channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. Results. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. Conclusions. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology. PMID:21508103

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

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

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji

2009-05-15

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carryingmore » humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.« less

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

PubMed Central

Malliaras, Konstantinos; Zhang, Yiqiang; Seinfeld, Jeffrey; Galang, Giselle; Tseliou, Eleni; Cheng, Ke; Sun, Baiming; Aminzadeh, Mohammad; Marbán, Eduardo

2013-01-01

Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation. PMID:23255322

Methylphenidate bioavailability in adults when an extended-release multiparticulate formulation is administered sprinkled on food or as an intact capsule.

PubMed

Pentikis, Helen S; Simmons, Roy D; Benedict, Michael F; Hatch, Simon J

2002-04-01

To determine the single-dose bioavailability of 20-mg Metadate CD (methylphenidate HCI, USP) Extended-Release Capsules sprinkled onto 1 level tablespoon (15 mL) of applesauce relative to an intact capsule under fasted conditions in healthy adults. This was a single-center, open-label, single-dose, randomized, two-way crossover study with a 6-day washout period between doses, in healthy male and female subjects (N= 26), aged 21-40 years. Plasma concentration-time data for methylphenidate were used to calculate the pharmacokinetic parameters for each treatment. The pharmacokinetic profile for Metadate CD exhibited biphasic release characteristics with a sharp initial slope and a second rising portion. For Cmax (maximum observed concentration), AUC(0-infinity) (area under the plasma concentration curve from time 0 to infinity) and AUC(0-infinity) (area under the plasma concentration curve from time 0 to the last measurable time point), the geometric least squares mean ratios and 90% confidence intervals were within the 80% to 125% confidence interval for bioequivalence. Adverse events were similar to those reported for methylphenidate. The bioavailability of methylphenidate was not altered when Metadate CD capsules were administered by sprinkling their contents onto a small amount of applesauce.

CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules

PubMed Central

Shamri, Revital; Melo, Rossana C. N.; Young, Kristen M.; Bivas-Benita, Maytal; Xenakis, Jason J.; Spencer, Lisa A.; Weller, Peter F.

2012-01-01

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC50 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.—Shamri, R., Melo, R. C. N., Young, K. M., B.-B, M., Xenakis, J. J., Spencer, L. A., Weller, P. F. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules. PMID:22294786

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

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

Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang

1988-01-01

A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with ({sup 14}C)chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of {sup 14}C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated {sup 14}C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intactmore » mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.« less

Plasticity of Intact Rubral Projections Mediates Spontaneous Recovery of Function after Corticospinal Tract Injury

PubMed Central

Siegel, Chad S.; Fink, Kathren L.; Strittmatter, Stephen M.

2015-01-01

Axons in the adult CNS fail to regenerate after injury, and therefore recovery from spinal cord injury (SCI) is limited. Although full recovery is rare, a modest degree of spontaneous recovery is observed consistently in a broad range of clinical and nonclinical situations. To define the mechanisms mediating spontaneous recovery of function after incomplete SCI, we created bilaterally complete medullary corticospinal tract lesions in adult mice, eliminating a crucial pathway for voluntary skilled movement. Anatomic and pharmacogenetic tools were used to identify the pathways driving spontaneous functional recovery in wild-type and plasticity-sensitized mice lacking Nogo receptor 1. We found that plasticity-sensitized mice recovered 50% of normal skilled locomotor function within 5 weeks of lesion. This significant, yet incomplete, spontaneous recovery was accompanied by extensive sprouting of intact rubrofugal and rubrospinal projections with the emergence of a de novo circuit between the red nucleus and the nucleus raphe magnus. Transient silencing of this rubro–raphe circuit in vivo via activation of the inhibitory DREADD (designer receptor exclusively activated by designer drugs) receptor hM4di abrogated spontaneous functional recovery. These data highlight the pivotal role of uninjured motor circuit plasticity in supporting functional recovery after trauma, and support a focus of experimental strategies on enhancing intact circuit rearrangement to promote functional recovery after SCI. PMID:25632122

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

Small Particles Intact Capture Experiment (SPICE)

NASA Technical Reports Server (NTRS)

Nishioka, Ken-Ji; Carle, G. C.; Bunch, T. E.; Mendez, David J.; Ryder, J. T.

1994-01-01

The Small Particles Intact Capture Experiment (SPICE) will develop technologies and engineering techniques necessary to capture nearly intact, uncontaminated cosmic and interplanetary dust particles (IDP's). Successful capture of such particles will benefit the exobiology and planetary science communities by providing particulate samples that may have survived unaltered since the formation of the solar system. Characterization of these particles may contribute fundamental data to our knowledge of how these particles could have formed into our planet Earth and, perhaps, contributed to the beginnings of life. The term 'uncontaminated' means that captured cosmic and IDP particles are free of organic contamination from the capture process and the term 'nearly intact capture' means that their chemical and elemental components are not materially altered during capture. The key to capturing cosmic and IDP particles that are organic-contamination free and nearly intact is the capture medium. Initial screening of capture media included organic foams, multiple thin foil layers, and aerogel (a silica gel); but, with the exception of aerogel, the requirements of no contamination or nearly intact capture were not met. To ensure no contamination of particles in the capture process, high-purity aerogel was chosen. High-purity aerogel results in high clarity (visual clearness), a useful quality in detection and recovery of embedded captured particles from the aerogel. P. Tsou at the Jet Propulsion Laboratory (JPL) originally described the use of aerogel for this purpose and reported laboratory test results. He has flown aerogel as a 'GAS-can Lid' payload on STS-47 and is evaluating the results. The Timeband Capture Cell Experiment (TICCE), a Eureca 1 experiment, is also flying aerogel and is scheduled for recovery in late April.

Magnetic targeting of nanoparticles across the intact blood–brain barrier

PubMed Central

Kong, Seong Deok; Lee, Jisook; Ramachandran, Srinivasan; Eliceiri, Brian P.; Shubayev, Veronica I.; Lal, Ratnesh; Jin, Sungho

2015-01-01

Delivery of therapeutic or diagnostic agents across an intact blood–brain barrier (BBB) remains a major challenge. Here we demonstrate in a mouse model that magnetic nanoparticles (MNPs) can cross the normal BBB when subjected to an external magnetic field. Following a systemic administration, an applied external magnetic field mediates the ability of MNPs to permeate the BBB and accumulate in a perivascular zone of the brain parenchyma. Direct tracking and localization inside endothelial cells and in the perivascular extracellular matrix in vivo was established using fluorescent MNPs. These MNPs were inert and associated with low toxicity, using a non-invasive reporter for astrogliosis, biochemical and histological studies. Atomic force microscopy demonstrated that MNPs were internalized by endothelial cells, suggesting that trans-cellular trafficking may be a mechanism for the MNP crossing of the BBB observed. The silica-coated magnetic nanocapsules (SiMNCs) allow on-demand drug release via remote radio frequency (RF) magnetic field. Together, these results establish an effective strategy for regulating the biodistribution of MNPs in the brain through the application of an external magnetic field. PMID:23063548

Assessing the use of immersive virtual reality, mouse and touchscreen in pointing and dragging-and-dropping tasks among young, middle-aged and older adults.

PubMed

Chen, Jiayin; Or, Calvin

2017-11-01

This study assessed the use of an immersive virtual reality (VR), a mouse and a touchscreen for one-directional pointing, multi-directional pointing, and dragging-and-dropping tasks involving targets of smaller and larger widths by young (n = 18; 18-30 years), middle-aged (n = 18; 40-55 years) and older adults (n = 18; 65-75 years). A three-way, mixed-factorial design was used for data collection. The dependent variables were the movement time required and the error rate. Our main findings were that the participants took more time and made more errors in using the VR input interface than in using the mouse or the touchscreen. This pattern applied in all three age groups in all tasks, except for multi-directional pointing with a larger target width among the older group. Overall, older adults took longer to complete the tasks and made more errors than young or middle-aged adults. Larger target widths yielded shorter movement times and lower error rates in pointing tasks, but larger targets yielded higher rates of error in dragging-and-dropping tasks. Our study indicated that any other virtual environments that are similar to those we tested may be more suitable for displaying scenes than for manipulating objects that are small and require fine control. Although interacting with VR is relatively difficult, especially for older adults, there is still potential for older adults to adapt to that interface. Furthermore, adjusting the width of objects according to the type of manipulation required might be an effective way to promote performance. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analysis of 16S libraries of mouse gastrointestinal microflora reveals a large new group of mouse intestinal bacteria.

PubMed

Salzman, Nita H; de Jong, Hendrik; Paterson, Yvonne; Harmsen, Hermie J M; Welling, Gjalt W; Bos, Nicolaas A

2002-11-01

Total genomic DNA from samples of intact mouse small intestine, large intestine, caecum and faeces was used as template for PCR amplification of 16S rRNA gene sequences with conserved bacterial primers. Phylogenetic analysis of the amplification products revealed 40 unique 16S rDNA sequences. Of these sequences, 25% (10/40) corresponded to described intestinal organisms of the mouse, including Lactobacillus spp., Helicobacter spp., segmented filamentous bacteria and members of the altered Schaedler flora (ASF360, ASF361, ASF502 and ASF519); 75% (30/40) represented novel sequences. A large number (11/40) of the novel sequences revealed a new operational taxonomic unit (OTU) belonging to the Cytophaga-Flavobacter-Bacteroides phylum, which the authors named 'mouse intestinal bacteria'. 16S rRNA probes were developed for this new OTU. Upon analysis of the novel sequences, eight were found to cluster within the Eubacterium rectale-Clostridium coccoides group and three clustered within the Bacteroides group. One of the novel sequences was distantly related to Verrucomicrobium spinosum and one was distantly related to Bacillus mycoides. Oligonucleotide probes specific for the 16S rRNA of these novel clones were generated. Using a combination of four previously described and four newly designed probes, approximately 80% of bacteria recovered from the murine large intestine and 71% of bacteria recovered from the murine caecum could be identified by fluorescence in situ hybridization (FISH).

Feasible pickup from intact ossicular chain with floating piezoelectric microphone.

PubMed

Kang, Hou-Yong; Na, Gao; Chi, Fang-Lu; Jin, Kai; Pan, Tie-Zheng; Gao, Zhen

2012-02-22

Many microphones have been developed to meet with the implantable requirement of totally implantable cochlear implant (TICI). However, a biocompatible one without destroying the intactness of the ossicular chain still remains under investigation. Such an implantable floating piezoelectric microphone (FPM) has been manufactured and shows an efficient electroacoustic performance in vitro test at our lab. We examined whether it pick up sensitively from the intact ossicular chain and postulated whether it be an optimal implantable one. Animal controlled experiment: five adult cats (eight ears) were sacrificed as the model to test the electroacoustic performance of the FPM. Three groups were studied: (1) the experiment group (on malleus): the FPM glued onto the handle of the malleus of the intact ossicular chains; (2) negative control group (in vivo): the FPM only hung into the tympanic cavity; (3) positive control group (Hy-M30): a HiFi commercial microphone placed close to the site of the experiment ear. The testing speaker played pure tones orderly ranged from 0.25 to 8.0 kHz. The FPM inside the ear and the HiFi microphone simultaneously picked up acoustic vibration which recorded as .wav files to analyze. The FPM transformed acoustic vibration sensitively and flatly as did the in vitro test across the frequencies above 2.0 kHz, whereas inefficiently below 1.0 kHz for its overloading mass. Although the HiFi microphone presented more efficiently than the FPM did, there was no significant difference at 3.0 kHz and 8.0 kHz. It is feasible to develop such an implantable FPM for future TICIs and TIHAs system on condition that the improvement of Micro Electromechanical System and piezoelectric ceramic material technology would be applied to reduce its weight and minimize its size.

Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

PubMed

Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

2013-03-05

One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Rhythmic Ganglion Cell Activity in Bleached and Blind Adult Mouse Retinas

PubMed Central

Menzler, Jacob; Channappa, Lakshmi; Zeck, Guenther

2014-01-01

In retinitis pigmentosa – a degenerative disease which often leads to incurable blindness- the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor’s dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance <200 µm) reveals synchrony among homologous RGC types and a constant phase shift (∼70 msec) among heterologous cell types (ON versus OFF). The rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

PubMed Central

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

Layer-specific chromatin accessibility landscapes reveal regulatory networks in adult mouse visual cortex

PubMed Central

Gray, Lucas T; Yao, Zizhen; Nguyen, Thuc Nghi; Kim, Tae Kyung; Zeng, Hongkui; Tasic, Bosiljka

2017-01-01

Mammalian cortex is a laminar structure, with each layer composed of a characteristic set of cell types with different morphological, electrophysiological, and connectional properties. Here, we define chromatin accessibility landscapes of major, layer-specific excitatory classes of neurons, and compare them to each other and to inhibitory cortical neurons using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We identify a large number of layer-specific accessible sites, and significant association with genes that are expressed in specific cortical layers. Integration of these data with layer-specific transcriptomic profiles and transcription factor binding motifs enabled us to construct a regulatory network revealing potential key layer-specific regulators, including Cux1/2, Foxp2, Nfia, Pou3f2, and Rorb. This dataset is a valuable resource for identifying candidate layer-specific cis-regulatory elements in adult mouse cortex. DOI: http://dx.doi.org/10.7554/eLife.21883.001 PMID:28112643

Maternal choline supplementation programs greater activity of the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in adult Ts65Dn trisomic mice

PubMed Central

Yan, Jian; Ginsberg, Stephen D.; Powers, Brian; Alldred, Melissa J.; Saltzman, Arthur; Strupp, Barbara J.; Caudill, Marie A.

2014-01-01

Maternal choline supplementation (MCS) induces lifelong cognitive benefits in the Ts65Dn mouse, a trisomic mouse model of Down syndrome and Alzheimer's disease. To gain insight into the mechanisms underlying these beneficial effects, we conducted a study to test the hypothesis that MCS alters choline metabolism in adult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic (2N) female littermates born to choline-unsupplemented or choline-supplemented Ts65Dn dams. Enrichment of d9-choline metabolites (derived from intact choline) and d3 + d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethanolamine N-methyltransferase (PEMT)] was measured in harvested tissues. Adult offspring (both Ts65Dn and 2N) of choline-supplemented (vs. choline-unsupplemented) dams exhibited 60% greater (P≤0.007) activity of hepatic PEMT, which functions in de novo choline synthesis and produces phosphatidylcholine (PC) enriched in docosahexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver, plasma, and brain in both genotypes but to a greater extent in the Ts65Dn adult offspring. MCS also yielded higher (P<0.05) d9 metabolite enrichments in liver, plasma, and brain. These data demonstrate that MCS exerts lasting effects on offspring choline metabolism, including up-regulation of the hepatic PEMT pathway and enhanced provision of choline and PEMT-PC to the brain.—Yan, J., Ginsberg, S. D., Powers, B., Alldred, M. J., Saltzman, A., Strupp, B. J., Caudill, M. A. Maternal choline supplementation programs greater activity of the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in adult Ts65Dn trisomic mice. PMID:24963152

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.

PubMed

Ye, Xin; Smallwood, Philip; Nathans, Jeremy

2011-01-01

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. Copyright © 2010 Elsevier B.V. All rights reserved.

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain

PubMed Central

Ye, Xin; Smallwood, Philip; Nathans, Jeremy

2011-01-01

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. PMID:21055480

The Thoc1 Encoded Ribonucleoprotein Is Required for Myeloid Progenitor Cell Homeostasis in the Adult Mouse

PubMed Central

Chinnam, Meenalakshmi; Povinelli, Benjamin J.; Fisher, Daniel T.; Golding, Michelle; Appenheimer, Michelle M.; Nemeth, Michael J.; Evans, Sharon; Goodrich, David W.

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover. PMID:24830368

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

PubMed

Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

50 CFR 622.186 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

50 CFR 622.186 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish... specified in paragraphs (c) and (d) of this section. Such fish may be eviscerated, gilled, and scaled, but...

Pharmacokinetics of erythropoietin in intact and anephric dogs

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

Fu, J.S.; Lertora, J.J.; Brookins, J.

1988-06-01

The present studies were performed to determine the pharmacokinetic parameters of erythropoietin in intact and anephric dogs by use of unlabeled crude native erythropoietin (nEp) and iodine 125-labeled purified recombinant erythropoietin (rEp) given by intravenous infusion for 15 minutes. Sephadex G-75 gel filtration was used to confirm that the 125I-rEp molecule remained iodinated in dog plasma during the 24-hour period of these studies. The plasma disappearance of erythropoietin conformed to a biexponential equation for both nEp and 125I-rEp, with the central compartment being larger than the peripheral compartment. The mean distribution half-life of 75.3 +/- 21.2 minutes for nEp wasmore » significantly (p less than 0.05) longer than that of 125I-rEp (23.7 +/- 5.0 minutes) in intact dogs. The intercompartmental clearance (CIic) for nEp (0.018 +/- 0.006 L/kg/hr) was significantly smaller than that of 125I-rEp (0.068 +/- 0.018 L/kg/hr) in intact dogs (p less than 0.05). There were no significant differences in apparent volume of distribution, elimination half-life, and elimination clearance (CIe) for nEp and rEp in intact dogs. The mean elimination half-life for 125I-rEp in intact dogs (9.0 +/- 0.6 hours) and anephric dogs (13.8 +/- 1.4 hours) was significantly different (p less than 0.05). The CIe for 125I-rEp in anephric dogs (0.008 +/- 0.001 L/kg/hr) was significantly (p less than 0.05) smaller than that of 125I-rEp in intact dogs (0.011 +/- 0.001 L/kg/hr). There were no significant differences in apparent volume of distribution, distribution half-life, and CIic for 125I-rEp in intact and anephric dogs.« less

Automated cell-type classification in intact tissues by single-cell molecular profiling

PubMed Central

2018-01-01

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research. PMID:29319504

p120 catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas

PubMed Central

Hendley, Audrey M.; Provost, Elayne; Bailey, Jennifer M.; Wang, Yue J.; Cleveland, Megan H.; Blake, Danielle; Bittman, Ross W.; Roeser, Jeffrey C.; Maitra, Anirban; Reynolds, Albert B.; Leach, Steven D.

2015-01-01

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120f/f pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. PMID:25523391

Mouse genetic differences in voluntary wheel running, adult hippocampal neurogenesis and learning on the multi-strain-adapted plus water maze

PubMed Central

Merritt, Jennifer; Rhodes, Justin S.

2014-01-01

Moderate levels of aerobic exercise broadly enhance cognition throughout the lifespan. One hypothesized contributing mechanism is increased adult hippocampal neurogenesis. Recently, we measured the effects of voluntary wheel running on adult hippocampal neurogenesis in 12 different mouse strains, and found increased neurogenesis in all strains, ranging from 2 to 5 fold depending on the strain. The purpose of this study was to determine the extent to which increased neurogenesis from wheel running is associated with enhanced performance on the water maze for 5 of the 12 strains, chosen based on their levels of neurogenesis observed in the previous study (C57BL/6J, 129S1/SvImJ, B6129SF1/J, DBA/2J, and B6D2F1/J). Mice were housed with or without a running wheels for 30 days then tested for learning and memory on the plus water maze, adapted for multiple strains, and rotarod test of motor performance. The first 10 days, animals were injected with BrdU to label dividing cells. After behavioral testing animals were euthanized to measure adult hippocampal neurogenesis using standard methods. Levels of neurogenesis depended on strain but all mice had a similar increase in neurogenesis in response to exercise. All mice acquired the water maze but performance depended on strain. Exercise improved water maze performance in all strains to a similar degree. Rotarod performance depended on strain. Exercise improved rotarod performance only in DBA/2J and B6D2F1/J mice. Taken together, results demonstrate that despite different levels of neurogenesis, memory performance and motor coordination in these mouse strains, all strains have the capacity to increase neurogenesis and improve learning on the water maze through voluntary wheel running. PMID:25435316

Vestibular dysfunction in the adult CBA/CaJ mouse after lead and cadmium treatment

PubMed Central

Klimpel, Katarina E. M.; Lee, Min Young; King, W. Michael; Raphael, Yehoash; Schacht, Jochen; Neitzel, Richard L.

2017-01-01

OBJECTIVES The vestibular system allows the perception of position and motion and its dysfunction presents as motion impairment, vertigo and balance abnormalities, leading to debilitating psychological discomfort and difficulty performing daily tasks. Although declines and deficits in vestibular function have been noted in rats exposed to lead (Pb) and in humans exposed to Pb and cadmium (Cd), no studies have directly examined the pathological and pathophysiological effects upon the vestibular apparatus of the inner ear. METHODS Eighteen young adult mice were exposed through their drinking water (3 mM Pb, 300 μM Cd, or a control treatment) for 10 weeks. Before and after treatment, they underwent a vestibular assessment, consisting of a rotarod performance test and a novel head stability test to measure the vestibulocolic reflex. At the conclusion of the study, the utricles were analyzed immunohistologically for condition of hair cells and nerve fibers. RESULTS Increased levels of Pb exposure correlated with decreased head stability in space; no significant decline in performance on rotarod test was found. No damage to the hair cells or the nerve fibers of the utricle was observed in histology. CONCLUSIONS The young adult CBA/CaJ mouse is able to tolerate occupationally-relevant Pb and Cd exposure well, but the correlation between Pb exposure and reduced head stability suggests that Pb exposure causes a decline in vestibular function. PMID:27257108

Leaky RAG Deficiency in Adult Patients with Impaired Antibody Production against Bacterial Polysaccharide Antigens

PubMed Central

Geier, Christoph B.; Piller, Alexander; Linder, Angela; Sauerwein, Kai M. T.; Eibl, Martha M.; Wolf, Hermann M.

2015-01-01

Loss of function mutations in the recombination activating genes RAG1 and RAG2 have been reported to cause a T-B-NK+ type of severe combined immunodeficiency. In addition identification of hypomorphic mutations in RAG1 and RAG2 has led to an expansion of the spectrum of disease to include Omenn syndrome, early onset autoimmunity, granuloma, chronic cytomegalovirus- or EBV-infection with expansion of gamma/delta T-cells, idiophatic CD4 lymphopenia and a phenotype resembling common variable immunodeficiency. Herein we describe a novel presentation of leaky RAG1 and RAG2 deficiency in two unrelated adult patients with impaired antibody production against bacterial polysaccharide antigens. Clinical manifestation included recurrent pneumonia, sinusitis, otitis media and in one patient recurrent cutaneous vasculitis. Both patients harbored a combination of a null mutation on one allele with a novel hypomorphic RAG1/2 mutation on the other allele. One of these novel mutations affected the start codon of RAG1 and resulted in an aberrant gene and protein expression. The second novel RAG2 mutation leads to a truncated RAG2 protein, lacking the C-terminus with intact core RAG2 and reduced VDJ recombination capacity as previously described in a mouse model. Both patients presented with severely decreased numbers of naïve CD4+ T cells and defective T independent IgG responses to bacterial polysaccharide antigens, while T cell-dependent IgG antibody formation e.g. after tetanus or TBEV vaccination was intact. In conclusion, hypomorphic mutations in genes responsible for SCID should be considered in adults with predominantly antibody deficiency. PMID:26186701

Fast synchronized dual-wavelength laser speckle imaging system for monitoring hemodynamic changes in a stroke mouse model

PubMed Central

Qin, Jia; Shi, Lei; Dziennis, Suzan; Reif, Roberto; Wang, Ruikang K.

2014-01-01

In this paper, we describe a newly developed synchronized dual-wavelength laser speckle contrast imaging (SDW-LSCI) system, which contains two cameras that are synchronously triggered to acquire data. The system can acquire data at a high spatiotemporal resolution (up to 500Hz for ~1000×1000 pixels). A mouse model of stroke is used to demonstrate the capability for imaging the fast changes (within tens of milliseconds) in oxygenated and deoxygenated hemoglobin concentration, and the relative changes in blood flow in the mouse brain, through an intact cranium. This novel imaging technology will enable the study of fast hemodynamics and metabolic changes in vascular diseases. PMID:23027260

In vivo measurement of muscle output in intact Drosophila.

PubMed

Elliott, Christopher J H; Sparrow, John C

2012-01-01

We describe our methods for analysing muscle function in a whole intact small insect, taking advantage of a simple flexible optical beam to produce an inexpensive transducer with wide application. We review our previous data measuring the response to a single action potential driven muscle twitch to explore jumping behaviour in Drosophila melanogaster. In the fruitfly, where the sophisticated and powerful genetic toolbox is being widely employed to investigate neuromuscular function, we further demonstrate the use of the apparatus to analyse in detail, within whole flies, neuronal and muscle mutations affecting activation of muscle contraction in the jump muscle. We have now extended the use of the apparatus to record the muscle forces during larval and other aspects of adult locomotion. The robustness, simplicity and versatility of the apparatus are key to these measurements. Copyright © 2011 Elsevier Inc. All rights reserved.

46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

Code of Federal Regulations, 2014 CFR

2014-10-01

... simplified stability proof test detailed in § 178.330 of this part, in the presence of a Coast Guard marine... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability requirements-monohull sailing vessels... PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178...

46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

Code of Federal Regulations, 2012 CFR

2012-10-01

... simplified stability proof test detailed in § 178.330 of this part, in the presence of a Coast Guard marine... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability requirements-monohull sailing vessels... PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178...

46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

Code of Federal Regulations, 2013 CFR

2013-10-01

... simplified stability proof test detailed in § 178.330 of this part, in the presence of a Coast Guard marine... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements-monohull sailing vessels... PASSENGER VESSELS (UNDER 100 GROSS TONS) INTACT STABILITY AND SEAWORTHINESS Intact Stability Standards § 178...

NIH Mouse Metabolic Phenotyping Centers: the power of centralized phenotyping.

PubMed

Laughlin, Maren R; Lloyd, K C Kent; Cline, Gary W; Wasserman, David H

2012-10-01

The Mouse Metabolic Phenotyping Centers (MMPCs) were founded in 2001 by the National Institutes of Health (NIH) to advance biomedical research by providing the scientific community with standardized, high-quality phenotyping services for mouse models of diabetes, obesity, and their complications. The intent is to allow researchers to take optimum advantage of the many new mouse models produced in labs and in high-throughput public efforts. The six MMPCs are located at universities around the country and perform complex metabolic tests in intact mice and hormone and analyte assays in tissues on a fee-for-service basis. Testing is subsidized by the NIH in order to reduce the barriers for mouse researchers. Although data derived from these tests belong to the researcher submitting mice or tissues, these data are archived after publication in a public database run by the MMPC Coordinating and Bioinformatics Unit. It is hoped that data from experiments performed in many mouse models of metabolic diseases, using standard protocols, will be useful in understanding the nature of these complex disorders. The current areas of expertise include energy balance and body composition, insulin action and secretion, whole-body and tissue carbohydrate and lipid metabolism, cardiovascular and renal function, and metabolic pathway kinetics. In addition to providing services, the MMPC staff provides expertise and advice to researchers, and works to develop and refine test protocols to best meet the community's needs in light of current scientific developments. Test technology is disseminated by publications and through annual courses.

A Pharmacokinetic Study Comparing Eslicarbazepine Acetate Administered Orally as a Crushed or Intact Tablet in Healthy Volunteers.

PubMed

Sunkaraneni, Soujanya; Kharidia, Jahnavi; Schutz, Ralph; Blum, David; Cheng, Hailong

2016-07-01

The relative bioequivalence of crushed versus intact eslicarbazepine acetate (ESL) tablets (800 mg) administered orally in healthy adults was evaluated in an open-label, randomized, 2-period crossover study with a 5-day washout between treatments. Sample blood levels of eslicarbazepine and (R)-licarbazepine were determined; pharmacokinetic parameters were derived for eslicarbazepine. Bioequivalence was established if the 90% confidence intervals (CIs) for the geometric mean treatment ratios of eslicarbazepine AUC(0-∞) and Cmax were within the prespecified 80%-125% range. Twenty-seven subjects in the intent-to-treat population (n = 28) completed both treatment periods. Eslicarbazepine exposure measures were similar for crushed versus intact ESL tablets: average Cmax , 11 700 versus 11 500 ng/mL; AUC(0-∞) , 225 000 versus 234 000 ng·h/mL; AUC(0-last) , 222 000 versus 231 000 ng·h/mL, respectively. Geometric least squares mean ratios (90%CIs) comparing eslicarbazepine exposure measures were within the 80%-125% range (Cmax , 102.63% [97.07%-108.51%]; AUC(0-∞) , 96.72% [94.36%-99.13%]; AUC0-last , 96.69% [94.24%-99.21%]). In conclusion, ESL administered orally as a crushed tablet sprinkled on applesauce, or intact were bioequivalent in healthy subjects. Eslicarbazepine bioavailability was not significantly altered by crushing, indicating that ESL tablets can be administered intact or crushed. © 2016, The American College of Clinical Pharmacology.

MiR-34a Regulates Axonal Growth of Dorsal Root Ganglia Neurons by Targeting FOXP2 and VAT1 in Postnatal and Adult Mouse.

PubMed

Jia, Longfei; Chopp, Michael; Wang, Lei; Lu, Xuerong; Zhang, Yi; Szalad, Alexandra; Zhang, Zheng Gang

2018-04-10

Hyperglycemia impairs nerve fibers of dorsal root ganglia (DRG) neurons, leading to diabetic peripheral neuropathy (DPN). However, the molecular mechanisms underlying DPN are not fully understood. Using a mouse model of type II diabetes (db/db mouse), we found that microRNA-34a (miR-34a) was over-expressed in DRG, sciatic nerve, and foot pad tissues of db/db mice. In vitro, high glucose significantly upregulated miR-34a in postnatal and adult DRG neurons, which was associated with inhibition of axonal growth. Overexpression and attenuation of miR-34a in postnatal and adult DRG neurons suppressed and promoted, respectively, axonal growth. Bioinformatic analysis suggested that miR-34a putatively targets forkhead box protein P2 (FOXP2) and vesicle amine transport 1 (VAT1), which were decreased in diabetic tissues and in cultured DRG neurons under high glucose conditions. Dual-luciferase assay showed that miR-34a downregulated FOXP2 and VAT1 expression by targeting their 3' UTR. Gain-of- and loss-of-function analysis showed an inverse relation between augmentation of miR-34a and reduction of FOXP2 and VAT1 proteins in postnatal and adult DRG neurons. Knockdown of FOXP2 and VAT1 reduced axonal growth. Together, these findings suggest that miR-34a and its target genes of FOXP2 and VAT1 are involved in DRG neuron damage under hyperglycemia.

Neuronal Representation of Ultraviolet Visual Stimuli in Mouse Primary Visual Cortex

PubMed Central

Tan, Zhongchao; Sun, Wenzhi; Chen, Tsai-Wen; Kim, Douglas; Ji, Na

2015-01-01

The mouse has become an important model for understanding the neural basis of visual perception. Although it has long been known that mouse lens transmits ultraviolet (UV) light and mouse opsins have absorption in the UV band, little is known about how UV visual information is processed in the mouse brain. Using a custom UV stimulation system and in vivo calcium imaging, we characterized the feature selectivity of layer 2/3 neurons in mouse primary visual cortex (V1). In adult mice, a comparable percentage of the neuronal population responds to UV and visible stimuli, with similar pattern selectivity and receptive field properties. In young mice, the orientation selectivity for UV stimuli increased steadily during development, but not direction selectivity. Our results suggest that, by expanding the spectral window through which the mouse can acquire visual information, UV sensitivity provides an important component for mouse vision. PMID:26219604

Deslorelin Implant Treatment for Hair Cycle Arrest (Alopecia X) in Two Intact Male Keeshonden.

PubMed

Layne, Elizabeth A; Richmond, Renee V

Alopecia in dogs occurs secondary to a variety of underlying inflammatory and noninflammatory conditions. Hair cycle arrest (alopecia X) is a noninflammatory alopecia that is frustrating to diagnose and treat due to lack of understanding of disease pathogenesis. A variety of therapies for hair cycle arrest have been described with inconsistent efficacy in different dog breeds and sexes; no definitive treatment is available. This report describes the use of a deslorelin acetate implant in two sexually intact adult male keeshonden, both diagnosed with hair cycle arrest. The dogs had progressive alopecia of the trunk that spared the head and distal limbs present for at least 2 yr. Diagnosis of hair cycle arrest was made based on clinical features, dermatohistopathology, and lack of systemic abnormalities. Treatment with a single subcutaneous 4.7 mg deslorelin acetate implant resulted in profuse hair regrowth within 3.5 mo that endured for at least 14 mo. Deslorelin implantation is a management option that is safe, affordable, and appears effective for sexually intact male keeshonden with hair cycle arrest.

50 CFR 622.10 - Landing fish intact--general.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact--general. 622.10... ATLANTIC General Provisions § 622.10 Landing fish intact—-general. This section contains requirements for landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified...

50 CFR 622.10 - Landing fish intact--general.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact--general. 622.10... ATLANTIC General Provisions § 622.10 Landing fish intact—-general. This section contains requirements for landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified...

The mouse-human anatomy ontology mapping project.

PubMed

Hayamizu, Terry F; de Coronado, Sherri; Fragoso, Gilberto; Sioutos, Nicholas; Kadin, James A; Ringwald, Martin

2012-01-01

The overall objective of the Mouse-Human Anatomy Project (MHAP) was to facilitate the mapping and harmonization of anatomical terms used for mouse and human models by Mouse Genome Informatics (MGI) and the National Cancer Institute (NCI). The anatomy resources designated for this study were the Adult Mouse Anatomy (MA) ontology and the set of anatomy concepts contained in the NCI Thesaurus (NCIt). Several methods and software tools were identified and evaluated, then used to conduct an in-depth comparative analysis of the anatomy ontologies. Matches between mouse and human anatomy terms were determined and validated, resulting in a highly curated set of mappings between the two ontologies that has been used by other resources. These mappings will enable linking of data from mouse and human. As the anatomy ontologies have been expanded and refined, the mappings have been updated accordingly. Insights are presented into the overall process of comparing and mapping between ontologies, which may prove useful for further comparative analyses and ontology mapping efforts, especially those involving anatomy ontologies. Finally, issues concerning further development of the ontologies, updates to the mapping files, and possible additional applications and significance were considered. DATABASE URL: http://obofoundry.org/cgi-bin/detail.cgi?id=ma2ncit.

The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

PubMed Central

Neal, Alice; Boldrin, Luisa; Morgan, Jennifer Elizabeth

2012-01-01

Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. PMID:22662253

Differential Regenerative Capacity of Neonatal Mouse Hearts after Cryoinjury

PubMed Central

Darehzereshki, Ali; Rubin, Nicole; Gamba, Laurent; Kim, Jieun; Fraser, James; Huang, Ying; Billings, Joshua; Mohammadzadeh, Robabeh; Wood, John; Warburton, David; Kaartinen, Vesa; Lien, Ching-Ling

2015-01-01

Neonatal mouse hearts fully regenerate after ventricular resection similar to adult zebrafish. We established cryoinjury models to determine if different types and varying degrees of severity in cardiac injuries trigger different responses in neonatal mouse hearts. In contrast to ventricular resection, neonatal mouse hearts fail to regenerate and show severe impairment of cardiac function post transmural cryoinjury. However, neonatal hearts fully recover after non-transmural cryoinjury. Interestingly, cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries. Epicardial activation and new coronary vessel formation occur after cryoinjury. The profibrotic marker PAI-1 is highly expressed after transmural but not non-transmural cryoinjuries, which may contribute to the differential scarring. Our results suggest that regenerative medicine strategies for heart injuries should vary depending on the nature of the injury. PMID:25555840

Neuroanatomical phenotyping of the mouse brain with three-dimensional autofluorescence imaging

PubMed Central

Wong, Michael D.; Dazai, Jun; Altaf, Maliha; Mark Henkelman, R.; Lerch, Jason P.; Nieman, Brian J.

2012-01-01

The structural organization of the brain is important for normal brain function and is critical to understand in order to evaluate changes that occur during disease processes. Three-dimensional (3D) imaging of the mouse brain is necessary to appreciate the spatial context of structures within the brain. In addition, the small scale of many brain structures necessitates resolution at the ∼10 μm scale. 3D optical imaging techniques, such as optical projection tomography (OPT), have the ability to image intact large specimens (1 cm3) with ∼5 μm resolution. In this work we assessed the potential of autofluorescence optical imaging methods, and specifically OPT, for phenotyping the mouse brain. We found that both specimen size and fixation methods affected the quality of the OPT image. Based on these findings we developed a specimen preparation method to improve the images. Using this method we assessed the potential of optical imaging for phenotyping. Phenotypic differences between wild-type male and female mice were quantified using computer-automated methods. We found that optical imaging of the endogenous autofluorescence in the mouse brain allows for 3D characterization of neuroanatomy and detailed analysis of brain phenotypes. This will be a powerful tool for understanding mouse models of disease and development and is a technology that fits easily within the workflow of biology and neuroscience labs. PMID:22718750

Palm is expressed in both developing and adult mouse lens and retina

PubMed Central

Castellini, Meryl; Wolf, Louise V; Chauhan, Bharesh K; Galileo, Deni S; Kilimann, Manfred W; Cvekl, Ales; Duncan, Melinda K

2005-01-01

Background Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. Methods The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. Results In the lens, Palm is already expressed at 9.5 dpc in the lens placode, and this expression is maintained in the lens vesicle throughout the formation of the adult lens. Palm is largely absent from the optic vesicle but is detectable at 10.5 dpc in the optic cup. In the developing retina, Palm expression transiently upregulates during the formation of optic nerve as well as in the formation of both the inner and outer plexiform layers. In short term dissociated chick retinal cultures, Palm protein is easily detectable, but the levels appear to reduce sharply as the cultures age. Palm mRNA was found at much higher levels relative to Palm2 or PalmD in both the retina and lens. Conclusion Palm is the major paralemmin family member expressed in the retina and lens and its expression in the retina transiently upregulates during active neurite outgrowth. The expression pattern of Palm in the eye is consistent with it being a Pax6 responsive gene. Since Palm is known to be able to drive membrane formation in brain neurons, it is possible that this molecule is crucial for the increase in membrane formation during lens fiber cell differentiation. PMID:15969763

Regional localization of activin-βA, activin-βC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

PubMed

Gold, Elspeth; Zellhuber-McMillan, Sylvia; Risbridger, Gail; Marino, Francesco Elia

2017-01-01

Activins and inhibins, members of the TGF-β superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-β A subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-β superfamily, the activin-β C subunit, as a novel antagonist of activin-A. Over-expression of activin-C (β C -β C ) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery of cancer biomarkers through the use of mouse models.

PubMed

Kuick, Rork; Misek, David E; Monsma, David J; Webb, Craig P; Wang, Hong; Peterson, Kelli J; Pisano, Michael; Omenn, Gilbert S; Hanash, Samir M

2007-04-28

Although our understanding of the molecular pathogenesis of common types of cancer has improved considerably, the development of effective strategies for cancer diagnosis and treatment have lagged behind. Mouse models of cancer potentially represent an efficient means for uncovering diagnostic markers as genetic alterations associated with human tumors can be engineered in mice. In addition, defined stages of tumor development, breeding conditions, and blood sampling can all be controlled and standardized to limit heterogeneity. Alternatively human cancer cells can be injected into mice and tumor development monitored in xenotransplants. Mouse-based studies promise to elucidate a repertoire of protein changes that occur in blood and biological fluids during tumor development. This is illustrated in a study in which we have applied a three-dimensional intact protein analysis system (IPAS) to elucidate detectable protein changes in serum from immunodeficient mice with lung xenografts from orthotopically implanted human A549 lung adenocarcinoma cells. With sufficiently detailed protein sequence identifications, the observed protein changes can be attributed to either the host mouse or the human tumor cells. It is noteworthy that the majority of increases identified have corresponded to relatively abundant serum proteins, some of which have previously been reported as increased in the sera of cancer patients. Proteomic studies of mouse models of cancer allow assessment of the range of changes in plasma proteins that occur with tumor development and may lead to the identification of potential cancer markers applicable to humans.

Olfactory discrimination training up-regulates and reorganizes expression of microRNAs in adult mouse hippocampus.

PubMed

Smalheiser, Neil R; Lugli, Giovanni; Lenon, Angela L; Davis, John M; Torvik, Vetle I; Larson, John

2010-02-26

Adult male mice (strain C57Bl/6J) were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour) or pseudo-training (exposed to two odours with reward not contingent upon response). These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct responses in 20 trials, occurring after three sessions (a total of approximately 40 min of training). The hippocampus was dissected bilaterally from each mouse (N = 7 in each group) and profiling of 585 miRNAs (microRNAs) was carried out using multiplex RT-PCR (reverse transcription-PCR) plates. A significant global up-regulation of miRNA expression was observed in the discrimination training versus pseudo-training comparison; when tested individually, 29 miRNAs achieved significance at P = 0.05. miR-10a showed a 2.7-fold increase with training, and is predicted to target several learning-related mRNAs including BDNF (brain-derived neurotrophic factor), CAMK2b (calcium/calmodulin-dependent protein kinase IIβ), CREB1 (cAMP-response-element-binding protein 1) and ELAVL2 [ELAV (embryonic lethal, abnormal vision, Drosophila)-like; Hu B]. Analysis of miRNA pairwise correlations revealed the existence of several miRNA co-expression modules that were specific to the training group. These in vivo results indicate that significant, dynamic and co-ordinated changes in miRNA expression accompany early stages of learning.

Chaotic electrical activity of living β-cells in the mouse pancreatic islet

NASA Astrophysics Data System (ADS)

Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

2007-02-01

To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

A physiologically based pharmacokinetic model for atrazine and its main metabolites in the adult male C57BL/6 mouse

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

Lin Zhoumeng; Interdisciplinary Toxicology Program, University of Georgia, Athens, GA 30602; Fisher, Jeffrey W.

Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR andmore » DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5 mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide.« less

Genomic structure, promoter identification, and chromosomal mapping of a mouse nuclear orphan receptor expressed in embryos and adult testes

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

Lee, C.H.; Wei, Li-Na; Copeland, N.G.

We have isolated and characterized overlapping genomic clones containing the complete transcribed region of a newly isolated mouse cDNA encoding an orphan receptor expressed specifically in midgestation embryos and adult testis. This gene spans a distance of more than 50 kb and is organized into 13 exons. The transcription initiation site is located at the 158th nucleotide upstream from the translation initiation codon. All the exon/intron junction sequences follow the GT/AG rule. Based upon Northern blot analysis and the size of the transcribed region of the gene, its transcript was determined to be approximately 2.5 kb. Within approximately 500 hpmore » upstream from the transcription initiation site, several immune response regulatory elements were identified but no TATA box was located. This gene was mapped to the distal region of mouse chromosome 10 and its locus has been designated Tr2-11. Immunohistochemical studies show that the Tr2-11 protein is present mainly in advanced germ cell populations of mature testes and that Tr2-11 gene expression is dramatically decreased in vitamin A-depleted animals. 23 refs., 7 figs.« less

p120 Catenin is required for normal tubulogenesis but not epithelial integrity in developing mouse pancreas.

PubMed

Hendley, Audrey M; Provost, Elayne; Bailey, Jennifer M; Wang, Yue J; Cleveland, Megan H; Blake, Danielle; Bittman, Ross W; Roeser, Jeffrey C; Maitra, Anirban; Reynolds, Albert B; Leach, Steven D

2015-03-01

The intracellular protein p120 catenin aids in maintenance of cell-cell adhesion by regulating E-cadherin stability in epithelial cells. In an effort to understand the biology of p120 catenin in pancreas development, we ablated p120 catenin in mouse pancreatic progenitor cells, which resulted in deletion of p120 catenin in all epithelial lineages of the developing mouse pancreas: islet, acinar, centroacinar, and ductal. Loss of p120 catenin resulted in formation of dilated epithelial tubules, expansion of ductal epithelia, loss of acinar cells, and the induction of pancreatic inflammation. Aberrant branching morphogenesis and tubulogenesis were also observed. Throughout development, the phenotype became more severe, ultimately resulting in an abnormal pancreas comprised primarily of duct-like epithelium expressing early progenitor markers. In pancreatic tissue lacking p120 catenin, overall epithelial architecture remained intact; however, actin cytoskeleton organization was disrupted, an observation associated with increased cytoplasmic PKCζ. Although we observed reduced expression of adherens junction proteins E-cadherin, β-catenin, and α-catenin, p120 catenin family members p0071, ARVCF, and δ-catenin remained present at cell membranes in homozygous p120(f/f) pancreases, potentially providing stability for maintenance of epithelial integrity during development. Adult mice homozygous for deletion of p120 catenin displayed dilated main pancreatic ducts, chronic pancreatitis, acinar to ductal metaplasia (ADM), and mucinous metaplasia that resembles PanIN1a. Taken together, our data demonstrate an essential role for p120 catenin in pancreas development. Copyright © 2014 Elsevier Inc. All rights reserved.

Autism Spectrum Disorder and intact executive functioning.

PubMed

Ferrara, R; Ansermet, F; Massoni, F; Petrone, L; Onofri, E; Ricci, P; Archer, T; Ricci, S

2016-01-01

Earliest notions concerning autism (Autism Spectrum Disorders, ASD) describe the disturbance in executive functioning. Despite altered definition, executive functioning, expressed as higher cognitive skills required complex behaviors linked to the prefrontal cortex, are defective in autism. Specific difficulties in children presenting autism or verbal disabilities at executive functioning levels have been identified. Nevertheless, the developmental deficit of executive functioning in autism is highly diversified with huge individual variation and may even be absent. The aim of the present study to examine the current standing of intact executive functioning intact in ASD. Analysis of ASD populations, whether high-functioning, Asperger's or autism Broad Phenotype, studied over a range of executive functions including response inhibition, planning, cognitive flexibility, cognitive inhibition, and alerting networks indicates an absence of damage/impairment compared to the typically-developed normal control subjects. These findings of intact executive functioning in ASD subjects provide a strong foundation on which to construct applications for growth environments and the rehabilitation of autistic subjects.

H3 and H4 Lysine Acetylation Correlates with Developmental and Experimentally Induced Adult Experience-Dependent Plasticity in the Mouse Visual Cortex

PubMed Central

Vierci, Gabriela; Pannunzio, Bruno; Bornia, Natalia; Rossi, Francesco M.

2016-01-01

Histone posttranslational modifications play a fundamental role in orchestrating gene expression. In this work, we analyzed the acetylation of H3 and H4 histones (AcH3–AcH4) and its modulation by visual experience in the mouse visual cortex (VC) during normal development and in two experimental conditions that restore juvenile-like plasticity levels in adults (fluoxetine treatment and enriched environment). We found that AcH3–AcH4 declines with age and is upregulated by treatments restoring plasticity in the adult. We also found that visual experience modulates AcH3–AcH4 in young and adult plasticity-restored mice but not in untreated ones. Finally, we showed that the transporter vGAT is downregulated in adult plasticity-restored models. In summary, we identified a dynamic regulation of AcH3–AcH4, which is associated with high plasticity levels and enhanced by visual experience. These data, along with recent ones, indicate H3–H4 acetylation as a central hub in the control of experience-dependent plasticity in the VC. PMID:27891053

μCT of ex-vivo stained mouse hearts and embryos enables a precise match between 3D virtual histology, classical histology and immunochemistry

PubMed Central

Larsson, Emanuel; Martin, Sabine; Lazzarini, Marcio; Tromba, Giuliana; Missbach-Guentner, Jeannine; Pinkert-Leetsch, Diana; Katschinski, Dörthe M.; Alves, Frauke

2017-01-01

The small size of the adult and developing mouse heart poses a great challenge for imaging in preclinical research. The aim of the study was to establish a phosphotungstic acid (PTA) ex-vivo staining approach that efficiently enhances the x-ray attenuation of soft-tissue to allow high resolution 3D visualization of mouse hearts by synchrotron radiation based μCT (SRμCT) and classical μCT. We demonstrate that SRμCT of PTA stained mouse hearts ex-vivo allows imaging of the cardiac atrium, ventricles, myocardium especially its fibre structure and vessel walls in great detail and furthermore enables the depiction of growth and anatomical changes during distinct developmental stages of hearts in mouse embryos. Our x-ray based virtual histology approach is not limited to SRμCT as it does not require monochromatic and/or coherent x-ray sources and even more importantly can be combined with conventional histological procedures. Furthermore, it permits volumetric measurements as we show for the assessment of the plaque volumes in the aortic valve region of mice from an ApoE-/- mouse model. Subsequent, Masson-Goldner trichrome staining of paraffin sections of PTA stained samples revealed intact collagen and muscle fibres and positive staining of CD31 on endothelial cells by immunohistochemistry illustrates that our approach does not prevent immunochemistry analysis. The feasibility to scan hearts already embedded in paraffin ensured a 100% correlation between virtual cut sections of the CT data sets and histological heart sections of the same sample and may allow in future guiding the cutting process to specific regions of interest. In summary, since our CT based virtual histology approach is a powerful tool for the 3D depiction of morphological alterations in hearts and embryos in high resolution and can be combined with classical histological analysis it may be used in preclinical research to unravel structural alterations of various heart diseases. PMID:28178293

Optical coherence elastography for cellular-scale stiffness imaging of mouse aorta

NASA Astrophysics Data System (ADS)

Wijesinghe, Philip; Johansen, Niloufer J.; Curatolo, Andrea; Sampson, David D.; Ganss, Ruth; Kennedy, Brendan F.

2017-04-01

We have developed a high-resolution optical coherence elastography system capable of estimating Young's modulus in tissue volumes with an isotropic resolution of 15 μm over a 1 mm lateral field of view and a 100 μm axial depth of field. We demonstrate our technique on healthy and hypertensive, freshly excised and intact mouse aortas. Our technique has the capacity to delineate the individual mechanics of elastic lamellae and vascular smooth muscle. Further, we observe global and regional vascular stiffening in hypertensive aortas, and note the presence of local micro-mechanical signatures, characteristic of fibrous and lipid-rich regions.

Maternal choline supplementation improves spatial learning and adult hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome

PubMed Central

Velazquez, Ramon; Ash, Jessica A.; Powers, Brian E.; Kelley, Christy M.; Strawderman, Myla; Luscher, Zoe I.; Ginsberg, Stephen D.; Mufson, Elliott J.; Strupp, Barbara J.

2014-01-01

In addition to intellectual disability, individuals with Down syndrome (DS) exhibit dementia by the third or fourth decade of life, due to the early onset of neuropathological changes typical of Alzheimer’s disease (AD). Deficient ontogenetic neurogenesis contributes to the brain hypoplasia and hypocellularity evident in fetuses and children with DS. A murine model of DS and AD (the Ts65Dn mouse) exhibits key features of these disorders, notably deficient ontogenetic neurogenesis, degeneration of basal forebrain cholinergic neurons (BFCNs), and cognitive deficits. Adult hippocampal (HP) neurogenesis is also deficient in Ts65Dn mice and may contribute to the observed cognitive dysfunction. Herein, we demonstrate that supplementing the maternal diet with additional choline (approximately 4.5 times the amount in normal rodent chow) dramatically improved the performance of the adult trisomic offspring in a radial arm water maze task. Ts65Dn offspring of choline-supplemented dams performed significantly better than unsupplemented Ts65Dn mice. Furthermore, adult hippocampal neurogenesis was partially normalized in the maternal choline supplemented (MCS) trisomic offspring relative to their unsupplemented counterparts. A significant correlation was observed between adult hippocampal neurogenesis and performance in the water maze, suggesting that the increased neurogenesis seen in the supplemented trisomic mice contributed functionally to their improved spatial cognition. These findings suggest that supplementing the maternal diet with additional choline has significant translational potential for DS. PMID:23643842

Altered expression of heat shock protein 110 family members in mouse hippocampal neurons following trimethyltin treatment in vivo and in vitro.

PubMed

Yoneyama, Masanori; Iwamoto, Naoko; Nagashima, Reiko; Sugiyama, Chie; Kawada, Koichi; Kuramoto, Nobuyuki; Shuto, Makoto; Ogita, Kiyokazu

2008-10-01

The heat shock protein (Hsp) 110 family is composed of HSP105, APG-1, and APG-2. As the response of these proteins to neuronal damage is not yet fully understood, in the present study, we assessed their expression in mouse hippocampal neurons following trimethyltin chloride (TMT) treatment in vivo and in vitro. Although each of these three Hsps had a distinct regional distribution within the hippocampus, a low level of all of them was observed in the granule cell layer of the dentate gyrus in naïve animals. TMT was effective in markedly increasing the level of these Hsps in the granule cell layer, at least 16h to 4days after the treatment. In the dentate granule cell layer on day 2 after TMT treatment, HSP105 was expressed mainly in the perikarya of NeuN-positive cells (intact neurons); whereas APG-1 and APG-2 were predominantly found in NeuN-negative cells (damaged neurons as evidenced by signs of cell shrinkage and condensation of chromatin). Assessments using primary cultures of mouse hippocampal neurons exposed to TMT revealed that whereas HSP105 was observed in intact neurons rather than in damaged neurons, APG-1 and APG-2 were detected in both damaged neurons and intact neurons. Taken together, our data suggest that APG-1 and APG-2 may play different roles from HSP105 in neurons damaged by TMT.

Acceleration of fusion in mouse palates by in vitro exposure to excess G

NASA Technical Reports Server (NTRS)

Duke, J.; Janer, L.; Campbell, M.

1984-01-01

Palatal shelves from 13- and 14-day mouse embryos were excised and cultured in contiguous pairs. Experimental cultures were exposed to 2.6 G in a culture centrifuge; controls were in the same incubator. After 24 hours, palates were prepared for light or electron microscopy. Scoring of paraffin sections according to the stage of fusion seen in the medial epithelial edges (MEE) showed that palates exposed to excess G were in more advanced stages of fusion than were controls. Ultrastructurally, control MEE had tightly apposed cell membranes and numerous desmosomes; in centrifuged MEE, desmosomes had been removed and there was much intercellular space. Nuclear membranes were intact in control MEE, but showed marked deterioration in MEE of centrifuged palates. Few lysosomes and no necrosis were seen in control MEE; centrifuged MEE had numerous lysosomes as well as necrotic cells. Basal lamina were intact in controls, but interrupted in centrifuged palates. The results confirm the hypothesis that gravitational increases speed up the differentiative process.

Two-photon laser scanning microscopy imaging of intact spinal cord and cerebral cortex reveals requirement for CXCR6 and neuroinflammation in immune cell infiltration of cortical injury sites.

PubMed

Kim, Jiyun V; Jiang, Ning; Tadokoro, Carlos E; Liu, Liping; Ransohoff, Richard M; Lafaille, Juan J; Dustin, Michael L

2010-01-31

The mouse spinal cord is an important site for autoimmune and injury models. Skull thinning surgery provides a minimally invasive window for microscopy of the mouse cerebral cortex, but there are no parallel methods for the spinal cord. We introduce a novel, facile and inexpensive method for two-photon laser scanning microscopy of the intact spinal cord in the mouse by taking advantage of the naturally accessible intervertebral space. These are powerful methods when combined with gene-targeted mice in which endogenous immune cells are labeled with green fluorescent protein (GFP). We first demonstrate that generation of the intervertebral window does not elicit a reaction of GFP(+) microglial cells in CX3CR1(gfp/+) mice. We next demonstrate a distinct rostrocaudal migration of GFP(+) immune cells in the spinal cord of CXCR6(gfp/+) mice during active experimental autoimmune encephalomyelitis (EAE). Interestingly, infiltration of the cerebral cortex by GFP(+) cells in these mice required three conditions: EAE induction, cortical injury and expression of CXCR6 on immune cells. Copyright 2009 Elsevier B.V. All rights reserved.

Adult-Derived Human Liver Stem/Progenitor Cells Infused 3 Days Postsurgery Improve Liver Regeneration in a Mouse Model of Extended Hepatectomy

PubMed Central

Herrero, Astrid; Prigent, Julie; Lombard, Catherine; Rosseels, Valérie; Daujat-Chavanieu, Martine; Breckpot, Karine; Najimi, Mustapha; Deblandre, Gisèle; Sokal, Etienne M.

2017-01-01

There is growing evidence that cell therapy constitutes a promising strategy for liver regenerative medicine. In the setting of hepatic cancer treatments, cell therapy could prove a useful therapeutic approach for managing the acute liver failure that occurs following extended hepatectomy. In this study, we examined the influence of delivering adult-derived human liver stem/progenitor cells (ADHLSCs) at two different early time points in an immunodeficient mouse model (Rag2−/-IL2Rg-/-) that had undergone a 70% hepatectomy procedure. The hepatic mesenchymal cells were intrasplenically infused either immediately after surgery (n = 26) or following a critical 3-day period (n = 26). We evaluated the cells' capacity to engraft at day 1 and day 7 following transplantation by means of human Alu qPCR quantification, along with histological assessment of human albumin and α-smooth muscle actin. In addition, cell proliferation (anti-mouse and human Ki-67 staining) and murine liver weight were measured in order to evaluate liver regeneration. At day 1 posttransplantation, the ratio of human to mouse cells was similar in both groups, whereas 1 week posttransplantation this ratio was significantly improved (p < 0.016) in mice receiving ADHLSC injection at day 3 posthepatectomy (1.7%), compared to those injected at the time of surgery (1%). On the basis of liver weight, mouse liver regeneration was more extensive 1 week posttransplantation in mice transplanted with ADHLSCs (+65.3%) compared to that of mice from the sham vehicle group (+42.7%). In conclusion, infusing ADHLSCs 3 days after extensive hepatectomy improves the cell engraftment and murine hepatic tissue regeneration, thereby confirming that ADHLSCs could be a promising cell source for liver cell therapy and hepatic tissue repair. PMID:27657746

Intact haptic priming in normal aging and Alzheimer's disease: evidence for dissociable memory systems.

PubMed

Ballesteros, Soledad; Reales, José Manuel

2004-01-01

This study is the first to report complete priming in Alzheimer's disease (AD) patients and older control subjects for objects presented haptically. To investigate possible dissociations between implicit and explicit objects representations, young adults, Alzheimer's patients, and older controls performed a speeded object naming task followed by a recognition task. Similar haptic priming was exhibited by the three groups, although young adults responded faster than the two older groups. Furthermore, there was no difference in performance between the two healthy groups. On the other hand, younger and older healthy adults did not differ on explicit recognition while, as expected, AD patients were highly impaired. The double dissociation suggests that different memory systems mediate both types of memory tasks. The preservation of intact haptic priming in AD provides strong support to the idea that object implicit memory is mediated by a memory system that is different from the medial-temporal diencephalic system underlying explicit memory, which is impaired early in AD. Recent imaging and behavioral studies suggest that the implicit memory system may depend on extrastriate areas of the occipital cortex although somatosensory cortical mechanisms may also be involved.

Cholesterol Depletion Disorganizes Oocyte Membrane Rafts Altering Mouse Fertilization

PubMed Central

Buschiazzo, Jorgelina; Ialy-Radio, Come; Auer, Jana; Wolf, Jean-Philippe; Serres, Catherine

2013-01-01

Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1) a decrease of the fertilization rate and index; and (2) a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol. PMID:23638166

Gender Differences in Performance of Script Analysis by Older Adults

ERIC Educational Resources Information Center

Helmes, E.; Bush, J. D.; Pike, D. L.; Drake, D. G.

2006-01-01

Script analysis as a test of executive functions is presumed sensitive to cognitive changes seen with increasing age. Two studies evaluated if gender differences exist in performance on scripts for familiar and unfamiliar tasks in groups of cognitively intact older adults. In Study 1, 26 older adults completed male and female stereotypical…

Childhood family disruptions and adult well-being: the differential effects of divorce and parental death.

PubMed

Mack, K Y

2001-01-01

This study draws on attachment theory and social learning theory and uses data from the National Survey of Families and Households to examine the differential effects of childhood family disruptions on adult well-being. Comparisons are made between adults who experienced parental divorce, adults who experienced parental death, and adults who were raised in intact families (N = 4,341). The present study differs from previous research by making direct comparisons between different family disruption groups, assessing the effects of family disruptions that occur before age 19, and including multiple measures of adult well-being as dependent variables. Consistent with hypotheses and inferences made from comparisons with adults from intact families, adults who experienced parental divorce report lower levels of parent-child relationship quality, higher levels of self-confidence, and lower levels of depression than adults who experienced parental death during childhood. Therefore, studies that fail to take type of childhood family disruption into account will lead to inaccurate and misleading conclusions about the effects of these experiences on adult outcomes.

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver.

PubMed

Grimm, Dirk; Wang, Lora; Lee, Joyce S; Schürmann, Nina; Gu, Shuo; Börner, Kathleen; Storm, Theresa A; Kay, Mark A

2010-09-01

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

Effect of human alpha 2HS glycoprotein on mouse macrophage function.

PubMed Central

Lewis, J G; André, C M

1980-01-01

alpha 2HS glycoprotein was isolated from normal adult serum. The ability of alpha 2HS glycoprotein to promote the endocytosis of radiolabelled DNA and radiolabelled latex particles by mouse macrophages was investigated. The results using both radiolabelled latex particles and radiolabelled DNA show that alpha 2HS glycoprotein enhances the ability of mouse macrophages to take up these radiolabelled substrates as compared to control cells. Images Figure 1 Figure 2 PMID:7439929

DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

PubMed Central

Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

2015-01-01

Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

Genetic disruption of ankyrin-G in adult mouse forebrain causes cortical synapse alteration and behavior reminiscent of bipolar disorder.

PubMed

Zhu, Shanshan; Cordner, Zachary A; Xiong, Jiali; Chiu, Chi-Tso; Artola, Arabiye; Zuo, Yanning; Nelson, Andrew D; Kim, Tae-Yeon; Zaika, Natalya; Woolums, Brian M; Hess, Evan J; Wang, Xiaofang; Chuang, De-Maw; Pletnikov, Mikhail M; Jenkins, Paul M; Tamashiro, Kellie L; Ross, Christopher A

2017-09-26

Genome-wide association studies have implicated the ANK3 locus in bipolar disorder, a major human psychotic illness. ANK3 encodes ankyrin-G, which organizes the neuronal axon initial segment (AIS). We generated a mouse model with conditional disruption of ANK3 in pyramidal neurons of the adult forebrain (Ank-G cKO). This resulted in the expected loss of pyramidal neuron AIS voltage-gated sodium and potassium channels. There was also dramatic loss of markers of afferent GABAergic cartridge synapses, resembling the cortical microcircuitry changes in brains from psychotic patients, and suggesting disinhibition. Expression of c-fos was increased in cortical pyramidal neurons, consistent with increased neuronal activity due to disinhibition. The mice showed robust behavioral phenotypes reminiscent of aspects of human mania, ameliorated by antimania drugs lithium and valproate. Repeated social defeat stress resulted in repeated episodes of dramatic behavioral changes from hyperactivity to "depression-like" behavior, suggestive of some aspects of human bipolar disorder. Overall, we suggest that this Ank-G cKO mouse model recapitulates some of the core features of human bipolar disorder and indicates that cortical microcircuitry alterations during adulthood may be involved in pathogenesis. The model may be useful for studying disease pathophysiology and for developing experimental therapeutics.

Glycogen synthase kinase-3 levels and phosphorylation undergo large fluctuations in mouse brain during development

PubMed Central

Beurel, Eléonore; Mines, Marjelo A; Song, Ling; Jope, Richard S

2012-01-01

Objectives Dysregulated glycogen synthase kinase-3 (GSK3) may contribute to the pathophysiology of mood disorders and other diseases, and appears to be a target of certain therapeutic drugs. The growing recognition of heightened vulnerability during development to many psychiatric diseases, including mood disorders, led us to test if there are developmental changes in mouse brain GSK3 and its regulation by phosphorylation and by therapeutic drugs. Methods GSK3 levels and phosphorylation were measured at seven ages of development in mouse cerebral cortex and hippocampus. Results Two periods of rapid transitions in GSK3 levels were identified, a large rise between postnatal day 1 and two to three weeks of age, where GSK3 levels were as high as four-fold adult mouse brain levels, and a rapid decline between two to four and eight weeks of age, when adult levels were reached. Inhibitory serine-phosphorylation of GSK3, particularly GSK3β, was extremely high in one-day postnatal mouse brain, and rapidly declined thereafter. These developmental changes in GSK3 were equivalent in male and female cerebral cortex, and differed from other signaling kinases, including Akt, ERK1/2, JNK, and p38 levels and phosphorylation. In contrast to adult mouse brain, where administration of lithium or fluoxetine rapidly and robustly increased serine-phosphorylation of GSK3, in young mice these responses were blunted or absent. Conclusions High brain levels of GSK3 and large fluctuations in its levels and phosphorylation in juvenile and adolescent mouse brain raise the possibility that they may contribute to destabilized mood regulation induced by environmental and genetic factors. PMID:23167932

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation.

PubMed

Zhang, Peng; Wu, Xinglong; Hu, Chunchao; Wang, Pengbo; Li, Xiangyun

2012-01-01

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.

Production of MPS VII mouse (Gustm(hE540A·mE536A)Sly) doubly tolerant to human and mouse β-glucuronidase

PubMed Central

Tomatsu, Shunji; Orii, Koji O.; Vogler, Carole; Grubb, Jeffrey H.; Snella, Elizabeth M.; Gutierrez, Monica; Dieter, Tatiana; Holden, Christopher C.; Sukegawa, Kazuko; Orii, Tadao; Kondo, Naomi; Sly, William S.

2006-01-01

Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by β-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5–26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. PMID:12700165

Somatic Cell Nuclear Transfer in the Mouse

NASA Astrophysics Data System (ADS)

Kishigami, Satoshi; Wakayama, Teruhiko

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

Maternal choline supplementation improves spatial learning and adult hippocampal neurogenesis in the Ts65Dn mouse model of Down syndrome.

PubMed

Velazquez, Ramon; Ash, Jessica A; Powers, Brian E; Kelley, Christy M; Strawderman, Myla; Luscher, Zoe I; Ginsberg, Stephen D; Mufson, Elliott J; Strupp, Barbara J

2013-10-01

In addition to intellectual disability, individuals with Down syndrome (DS) exhibit dementia by the third or fourth decade of life, due to the early onset of neuropathological changes typical of Alzheimer's disease (AD). Deficient ontogenetic neurogenesis contributes to the brain hypoplasia and hypocellularity evident in fetuses and children with DS. A murine model of DS and AD (the Ts65Dn mouse) exhibits key features of these disorders, notably deficient ontogenetic neurogenesis, degeneration of basal forebrain cholinergic neurons (BFCNs), and cognitive deficits. Adult hippocampal (HP) neurogenesis is also deficient in Ts65Dn mice and may contribute to the observed cognitive dysfunction. Herein, we demonstrate that supplementing the maternal diet with additional choline (approximately 4.5 times the amount in normal rodent chow) dramatically improved the performance of the adult trisomic offspring in a radial arm water maze task. Ts65Dn offspring of choline-supplemented dams performed significantly better than unsupplemented Ts65Dn mice. Furthermore, adult hippocampal neurogenesis was partially normalized in the maternal choline supplemented (MCS) trisomic offspring relative to their unsupplemented counterparts. A significant correlation was observed between adult hippocampal neurogenesis and performance in the water maze, suggesting that the increased neurogenesis seen in the supplemented trisomic mice contributed functionally to their improved spatial cognition. These findings suggest that supplementing the maternal diet with additional choline has significant translational potential for DS. Copyright © 2013 Elsevier Inc. All rights reserved.

Transitive inference in adults with autism spectrum disorders

PubMed Central

Solomon, Marjorie; Frank, Michael J.; Smith, Anne C.; Ly, Stanford; Carter, Cameron S.

2012-01-01

Individuals with autism spectrum disorders (ASDs) exhibit intact rote learning with impaired generalization. A transitive inference paradigm, involving training on four sequentially presented stimulus pairs containing overlapping items, with subsequent testing on two novel pairs, was used to investigate this pattern of learning in 27 young adults with ASDs and 31 matched neurotypical individuals (TYPs). On the basis of findings about memory and neuropathology, we hypothesized that individuals with ASDs would use a relational flexibility/conjunctive strategy reliant on an intact hippocampus, versus an associative strength/value transfer strategy requiring intact interactions between the prefrontal cortex and the striatum. Hypotheses were largely confirmed. ASDs demonstrated reduced interference from intervening pairs in early training; only TYPs formed a serial position curve by test; and ASDs exhibited impairments on the novel test pair consisting of end items with intact performance on the inner test pair. However, comparable serial position curves formed for both groups by the end of the first block. PMID:21656344

Cytoarchitecture of the spinal cord of the postnatal (P4) mouse.

PubMed

Sengul, Gulgun; Puchalski, Ralph B; Watson, Charles

2012-05-01

Interpretation of the new wealth of gene expression and molecular mechanisms in the developing mouse spinal cord requires an accurate anatomical base on which data can be mapped. Therefore, we have assembled a spinal cord atlas of the P4 mouse to facilitate direct comparison with the adult specimens and to contribute to studies of the development of the mouse spinal cord. This study presents the anatomy of the spinal cord of the P4 C57Bl/6J mouse using Nissl and acetyl cholinesterase-stained sections. It includes a detailed map of the laminar organization of selected spinal cord segments and a description of named cell groups of the spinal cord such as the central cervical (CeCv), lateral spinal nucleus, lateral cervical, and dorsal nuclei. The motor neuron groups have also been identified according to the muscle groups they are likely to supply. General features of Rexed's laminae of the P4 spinal cord showed similarities to that of the adult (P56). However, certain differences were observed with regard to the extent of laminae and location of certain cell groups, such as the dorsal nucleus having a more dispersed structure and a more ventral and medial position or the CeCv being located in the medial part of lamina 5 in contrast to the adult where it is located in lamina 7. Motor neuron pools appeared to be more tightly packed in the P4 spinal cord. The dorsal horn was relatively larger and there was more white matter in the P56 spinal cord. Copyright © 2012 Wiley Periodicals, Inc.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

PubMed

Trejter, Marcin; Jopek, Karol; Celichowski, Piotr; Tyczewska, Marianna; Malendowicz, Ludwik K; Rucinski, Marcin

2015-01-01

Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ m

Chronodependent effect of interleukin-2 on mouse spleen cells in the model of cyclophosphamide immunosuppression.

PubMed

Shurlygina, A V; Mel'nikova, E V; Trufakin, V A

2015-02-01

We studied the chronodependent effect of IL-2 in the experimental model of immunodeficiency, cyclophosphamide-induced immunosuppression in mice. IL-2 in a dose of 100 U/ mouse was administered at 10.00 and 16.00 for 3 days after injection of cyclophosphamide. In contrast to the morning treatment with the cytokine, evening administration produced antiapoptotic effect on splenocytes and stimulated proliferation to a greater extent. This was accompanied by an increase in the number of CD4(+), CD25(+) and CD4(+)25(+) cells in the spleen to a level of intact mice. More pronounced effect of the evening mode of IL-2 administration on the proliferation and subpopulation composition of mouse spleen cells in the studied model can be associated with high blood level of CD25(+) cells at this time of the day.

Less Daily Computer Use is Related to Smaller Hippocampal Volumes in Cognitively Intact Elderly.

PubMed

Silbert, Lisa C; Dodge, Hiroko H; Lahna, David; Promjunyakul, Nutta-On; Austin, Daniel; Mattek, Nora; Erten-Lyons, Deniz; Kaye, Jeffrey A

2016-01-01

Computer use is becoming a common activity in the daily life of older individuals and declines over time in those with mild cognitive impairment (MCI). The relationship between daily computer use (DCU) and imaging markers of neurodegeneration is unknown. The objective of this study was to examine the relationship between average DCU and volumetric markers of neurodegeneration on brain MRI. Cognitively intact volunteers enrolled in the Intelligent Systems for Assessing Aging Change study underwent MRI. Total in-home computer use per day was calculated using mouse movement detection and averaged over a one-month period surrounding the MRI. Spearman's rank order correlation (univariate analysis) and linear regression models (multivariate analysis) examined hippocampal, gray matter (GM), white matter hyperintensity (WMH), and ventricular cerebral spinal fluid (vCSF) volumes in relation to DCU. A voxel-based morphometry analysis identified relationships between regional GM density and DCU. Twenty-seven cognitively intact participants used their computer for 51.3 minutes per day on average. Less DCU was associated with smaller hippocampal volumes (r = 0.48, p = 0.01), but not total GM, WMH, or vCSF volumes. After adjusting for age, education, and gender, less DCU remained associated with smaller hippocampal volume (p = 0.01). Voxel-wise analysis demonstrated that less daily computer use was associated with decreased GM density in the bilateral hippocampi and temporal lobes. Less daily computer use is associated with smaller brain volume in regions that are integral to memory function and known to be involved early with Alzheimer's pathology and conversion to dementia. Continuous monitoring of daily computer use may detect signs of preclinical neurodegeneration in older individuals at risk for dementia.

Synchrotron phase-contrast X-ray imaging reveals fluid dosing dynamics for gene transfer into mouse airways.

PubMed

Donnelley, M; Siu, K K W; Jamison, R A; Parsons, D W

2012-01-01

Although airway gene transfer research in mouse models relies on bolus fluid dosing into the nose or trachea, the dynamics and immediate fate of delivered gene transfer agents are poorly understood. In particular, this is because there are no in vivo methods able to accurately visualize the movement of fluid in small airways of intact animals. Using synchrotron phase-contrast X-ray imaging, we show that the fate of surrogate fluid doses delivered into live mouse airways can now be accurately and non-invasively monitored with high spatial and temporal resolution. This new imaging approach can help explain the non-homogenous distributions of gene expression observed in nasal airway gene transfer studies, suggests that substantial dose losses may occur at deliver into mouse trachea via immediate retrograde fluid motion and shows the influence of the speed of bolus delivery on the relative targeting of conducting and deeper lung airways. These findings provide insight into some of the factors that can influence gene expression in vivo, and this method provides a new approach to documenting and analyzing dose delivery in small-animal models.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+ Sensitivity in Intact Cardiomyocytes

PubMed Central

Figueiredo-Freitas, Cícero; Dulce, Raul A.; Foster, Matthew W.; Liang, Jingsheng; Yamashita, Aline M.S.; Lima-Rosa, Frederico L.; Thompson, J. Will; Moseley, M. Arthur; Hare, Joshua M.; Nogueira, Leonardo; Sorenson, Martha M.

2015-01-01

Abstract Aims: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. Results: Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca2+ sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 μM GSNO also decreased Ca2+ sensitivity of contraction and 10 μM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥100 μM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca2+ sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography–tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10–500 μM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. Innovation and Conclusions: Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations. Antioxid. Redox Signal. 23, 1017–1034. PMID:26421519

In Vivo Axial Loading of the Mouse Tibia

PubMed Central

Melville, Katherine M.; Robling, Alexander G.

2015-01-01

Summary Non-invasive methods to apply controlled, cyclic loads to the living skeleton are used as an anabolic agent to stimulate new bone formation in adults and enhance bone mass accrual in growing animals. These methods are also invaluable for understanding bone signaling pathways. Our focus here is on a particular loading model: in vivo axial compression of the mouse tibia. An advantage of loading the tibia is that changes are present in both the cancellous envelope of the proximal tibia and the cortical bone of the tibial diaphysis. To load the tibia of the mouse axially in vivo, a cyclic compressive load is applied up to five times a week to a single tibia per mouse for a duration lasting from 1 day to 6 weeks. With the contralateral limb as an internal control, the anabolic response of the skeleton to mechanical stimuli can be studied in a pairwise experimental design. Here, we describe the key parameters that must be considered before beginning an in vivo mouse tibial loading experiment, including methods for in vivo strain gauging of the tibial midshaft, and then we describe general methods for loading the mouse tibia for an experiment lasting multiple days. PMID:25331046

Decreased adult hippocampal neurogenesis in the PDAPP mouse model of Alzheimer's disease.

PubMed

Donovan, Michael H; Yazdani, Umar; Norris, Rebekah D; Games, Dora; German, Dwight C; Eisch, Amelia J

2006-03-01

Abnormal subgranular zone (SGZ) neurogenesis is proposed to contribute to Alzheimer's disease (AD)-related decreases in hippocampal function. Our goal was to examine hippocampal neurogenesis in the PDAPP mouse, a model of AD with age-dependent accumulation of amyloid-beta(42) (Abeta(42))-containing plaques that is well studied with regard to AD therapies. A secondary goal was to determine whether altered neurogenesis in the PDAPP mouse is associated with abnormal maturation or number of mature cells. A tertiary goal was to provide insight into why hippocampal neurogenesis appears to be increased in AD post-mortem tissue and decreased in most AD mouse models. We report an age-dependent decrease in SGZ proliferation in homozygous PDAPP mice. At 1 year of age, PDAPP mice also had new dentate gyrus granule neurons with abnormal maturation and fewer dying cells relative to control mice. In contrast to decreased SGZ cell birth, PDAPP mice had increased birth of immature neurons in the outer portion of the granule cell layer (oGCL), providing insight into why some studies link AD with increased neurogenesis. However, these ectopic oGCL cells were still rare compared with SGZ proliferating cells, emphasizing that the primary characteristic of PDAPP mice is decreased neurogenesis. The decrease in SGZ neurogenesis was not associated with an age-dependent loss of dentate granule neurons. The altered neurogenesis in the PDAPP mouse may contribute to the age-related cognitive deficits reported in this model of AD and may be a useful adjunct target for assessing the impact of AD therapies. J. Comp. Neurol. 495:70-83, 2006. (c) 2006 Wiley-Liss, Inc.

Transepithelial SCFA fluxes link intracellular and extracellular pH regulation of mouse colonocytes.

PubMed

Chu, S; Montrose, M H

1997-10-01

We have studied pH regulation in both intracellular and extracellular compartments of mouse colonic crypts, using distal colonic mucosa with intact epithelial architecture. In this work, we question how transepithelial SCFA gradients affect intracellular pH (pHi) and examine interactions between extracellular pH (pHo) and pHi regulation in crypts of distal colonic epithelium from mouse. We studied pH regulation in three adjacent compartments of distal colonic epithelium (crypt lumen, crypt epithelial cell cytosol, and lamina propria) with SNARF-1 (a pH sensitive fluorescent dye), digital imaging microscopy (for pHi), and confocal microscopy (for pHo). Combining results from the three compartments allows us to find how pHi and pHo are regulated and related under the influence of physiological transepithelial SCFA gradients, and develop a better understanding of pH regulation mechanisms in colonic crypts. Results suggest a complex interdependency between SCFA fluxes and pHo values, which can directly affect how strongly SCFAs acidify colonocytes.

Altered protein prenylation in Sertoli cells is associated with adult infertility resulting from childhood mumps infection.

PubMed

Wang, Xiu-Xing; Ying, Pu; Diao, Fan; Wang, Qiang; Ye, Dan; Jiang, Chen; Shen, Ning; Xu, Na; Chen, Wei-Bo; Lai, Shan-Shan; Jiang, Shan; Miao, Xiao-Li; Feng, Jin; Tao, Wei-Wei; Zhao, Ning-Wei; Yao, Bing; Xu, Zhi-Peng; Sun, Hai-Xiang; Li, Jian-Min; Sha, Jia-Hao; Huang, Xing-Xu; Shi, Qing-Hua; Tang, Hong; Gao, Xiang; Li, Chao-Jun

2013-07-29

Mumps commonly affects children 5-9 yr of age, and can lead to permanent adult sterility in certain cases. However, the etiology of this long-term effect remains unclear. Mumps infection results in progressive degeneration of the seminiferous epithelium and, occasionally, Sertoli cell-only syndrome. Thus, the remaining Sertoli cells may be critical to spermatogenesis recovery after orchitis healing. Here, we report that the protein farnesylation/geranylgeranylation balance is critical for patients' fertility. The expression of geranylgeranyl diphosphate synthase 1 (GGPPS) was decreased due to elevated promoter methylation in the testes of infertile patients with mumps infection history. When we deleted GGPPS in mouse Sertoli cells, these cells remained intact, whereas the adjacent spermatogonia significantly decreased after the fifth postnatal day. The proinflammatory MAPK and NF-κB signaling pathways were constitutively activated in GGPPS(-/-) Sertoli cells due to the enhanced farnesylation of H-Ras. GGPPS(-/-) Sertoli cells secreted an array of cytokines to stimulate spermatogonia apoptosis, and chemokines to induce macrophage invasion into the seminiferous tubules. Invaded macrophages further blocked spermatogonia development, resulting in a long-term effect through to adulthood. Notably, this defect could be rescued by GGPP administration in EMCV-challenged mice. Our results suggest a novel mechanism by which mumps infection during childhood results in adult sterility.

46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this section...

46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-non-sailing vessels. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part, the...

A Novel ex vivo Mouse Mesometrium Culture Model for Investigating Angiogenesis in Microvascular Networks.

PubMed

Suarez-Martinez, Ariana D; Bierschenk, Susanne; Huang, Katie; Kaplan, Dana; Bayer, Carolyn L; Meadows, Stryder M; Sperandio, Markus; Murfee, Walter L

2018-05-18

The development of models that incorporate intact microvascular networks enables the investigation of multicellular dynamics during angiogenesis. Our laboratory introduced the rat mesentery culture model as such a tool, which would be enhanced with mouse tissue. Since mouse mesentery is avascular, an alternative is mouse mesometrium, the connective tissue of uterine horns. The study's objective was to demonstrate that mouse mesometrium contains microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Harvested mesometrium tissues from C57Bl/6 female mice were cultured in media with serum for up to 7 days. PECAM, NG2, αSMA, and LYVE-1 labeling identified endothelial cells, pericytes, smooth muscle cells, and lymphatic endothelial cells, respectively. These cells comprised microvascular networks with arterioles, venules, and capillaries. Compared to day 0, capillary sprouts per vascular length were increased by 3 and 5 days in culture (day 0, 0.08 ± 0.01; day 3, 3.19 ± 0.78; day 5, 2.49 ± 0.05 sprouts/mm; p < 0.05). Time-lapse imaging of cultured tissues from FlkEGFP mice showcases the use of the model for lineage studies. The impact is supported by the identification of endothelial cell jumping from one sprout to another. These results introduce a novel culture model for investigating multicellular dynamics during angiogenesis in real-time ex vivo microvascular networks. © 2018 S. Karger AG, Basel.

Intact unconscious processing of eye contact in schizophrenia.

PubMed

Seymour, Kiley; Rhodes, Gillian; Stein, Timo; Langdon, Robyn

2016-03-01

The perception of eye gaze is crucial for social interaction, providing essential information about another person's goals, intentions, and focus of attention. People with schizophrenia suffer a wide range of social cognitive deficits, including abnormalities in eye gaze perception. For instance, patients have shown an increased bias to misjudge averted gaze as being directed toward them. In this study we probed early unconscious mechanisms of gaze processing in schizophrenia using a technique known as continuous flash suppression. Previous research using this technique to render faces with direct and averted gaze initially invisible reveals that direct eye contact gains privileged access to conscious awareness in healthy adults. We found that patients, as with healthy control subjects, showed the same effect: faces with direct eye gaze became visible significantly faster than faces with averted gaze. This suggests that early unconscious processing of eye gaze is intact in schizophrenia and implies that any misjudgments of gaze direction must manifest at a later conscious stage of gaze processing where deficits and/or biases in attributing mental states to gaze and/or beliefs about being watched may play a role.

Later Life Parental Divorce and Widowhood: Impact on Young Adults' Assessment of Parent-Child Relations.

ERIC Educational Resources Information Center

Aquilino, William S.

1994-01-01

Explored implications of later life parental divorce and widowhood for relationship between parents and young adult children among 3,281 young adults who grew up in intact families. Family disruption that occurred after children were grown had sizable effects on parent-adult child relations, with later life divorce lowering relationship quality…

Retinoid-related orphan receptor γ (RORγ) adult induced knockout mice develop lymphoblastic lymphoma.

PubMed

Liljevald, Maria; Rehnberg, Maria; Söderberg, Magnus; Ramnegård, Marie; Börjesson, Jenny; Luciani, Donatella; Krutrök, Nina; Brändén, Lena; Johansson, Camilla; Xu, Xiufeng; Bjursell, Mikael; Sjögren, Anna-Karin; Hornberg, Jorrit; Andersson, Ulf; Keeling, David; Jirholt, Johan

2016-11-01

RORγ is a nuclear hormone receptor which controls polarization of naive CD4 + T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma. Copyright © 2016 Elsevier B.V. All rights reserved.

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

PubMed Central

Núñez, David; Comas, Laura; Lanuza, Pilar M.; Sánchez-Martinez, Diego; Pérez-Hernández, Marta; Catalán, Elena; Domingo, María Pilar; Velázquez-Campoy, Adrián; Pardo, Julián; Gálvez, Eva M.

2017-01-01

The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes. PMID:29312326

A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level.

PubMed

Núñez, David; Comas, Laura; Lanuza, Pilar M; Sánchez-Martinez, Diego; Pérez-Hernández, Marta; Catalán, Elena; Domingo, María Pilar; Velázquez-Campoy, Adrián; Pardo, Julián; Gálvez, Eva M

2017-01-01

The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver

PubMed Central

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao

2015-01-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

PubMed

Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

2015-12-01

Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

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

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua

2008-03-10

Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen genemore » expression were examined in CCN2{sup -/-} MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2{sup -/-} MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.« less

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PubMed

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

PubMed Central

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

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

Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.

Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressedmore » in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.« less

Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet; phenotype of the adult acetylcholinesterase deficient mouse.

PubMed

Duysen, Ellen G; Stribley, Judith A; Fry, Debra L; Hinrichs, Steven H; Lockridge, Oksana

2002-07-30

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE -/- mice were born alive and survived an average of 14 days. The emaciated appearance of AChE -/- mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (+/-) were fed 11% high fat chow supplemented with liquid Ensure. AChE -/- pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE -/- mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function. Copyright 2002 Elsevier Science B.V.

[Expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain].

PubMed

Zhang, Wei; Fan, Li-mei; Li, Lin-lin; Peng, Zheng-yu

2014-01-01

To investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in the development of mouse brain. Brain samples were collected from mice with different developmental stages: 9, 12, 14 d before birth (E9, E12, E14) and 1 d, 3 weeks and 3 months after birth. The expression of NSSR1 in mouse brain at different developmental stages was detected by Western blot and the distribution of NSSR1 was analyzed by immunohistochemical staining. The expression and distribution of NSSR1 in mouse brain were compared among embryos, neonatal and adult animals. During embryogenesis, the expression of NSSR1 proteins increases significantly from 0.186(E9) to 0.445(E14) and reached a high level after birth. Immunohistochemical analysis showed that in E12 embryos, NSSR1 was specifically distributed in the marginal and mantle layers. The expression of NSSR1 in hippocampus was very low in neonatal animals but stronger in adults. In cerebellar cortex, NSSR1 was widely expressed in purkinje and granule cells of adult animals, but mainly expressed in Purkinje cells in neonates. The expression of NSSR1 is regulated by the development of mouse brain and presents dynamic changes.

Facile synthesis of titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles for highly specific enrichment and analysis of intact phosphoproteins.

PubMed

Wang, Hao; Tian, Zhixin

2018-06-06

Analysis of phosphoproteins always faces the challenge of low stoichiometry, which demands highly selective and efficient enrichment in the initial sample preparation. Here we report our synthesis of the novel titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles (Ti 4+ -ATP-NPs) for efficient enrichment of intact phosphoproteins. The average diameter of Ti 4+ -ATP-NPs was about 128 nm with good dispersibility and the saturated adsorption capacity for β-casein was 1046.5 mg/g. In addition, Ti 4+ -ATP-NPs exhibited high specificity and selectivity in enriching phosphoproteins from both standard protein mixtures and complex biological samples (non-fat milk, chicken egg white and mouse heart tissue extract) as demonstrated by SDS-PAGE. Copyright © 2018 Elsevier B.V. All rights reserved.

Morphological phenotyping of mouse hearts using optical coherence tomography

NASA Astrophysics Data System (ADS)

Cua, Michelle; Lin, Eric; Lee, Ling; Sheng, Xiaoye; Wong, Kevin S. K.; Tibbits, Glen F.; Beg, Mirza Faisal; Sarunic, Marinko V.

2014-11-01

Transgenic mouse models have been instrumental in the elucidation of the molecular mechanisms behind many genetically based cardiovascular diseases such as Marfan syndrome (MFS). However, the characterization of their cardiac morphology has been hampered by the small size of the mouse heart. In this report, we adapted optical coherence tomography (OCT) for imaging fixed adult mouse hearts, and applied tools from computational anatomy to perform morphometric analyses. The hearts were first optically cleared and imaged from multiple perspectives. The acquired volumes were then corrected for refractive distortions, and registered and stitched together to form a single, high-resolution OCT volume of the whole heart. From this volume, various structures such as the valves and myofibril bundles were visualized. The volumetric nature of our dataset also allowed parameters such as wall thickness, ventricular wall masses, and luminal volumes to be extracted. Finally, we applied the entire acquisition and processing pipeline in a preliminary study comparing the cardiac morphology of wild-type mice and a transgenic mouse model of MFS.

Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells

PubMed Central

Zhang, Yiqiang; Zhong, Jiang F; Qiu, Hongyu; Robb MacLellan, W.; Marbán, Eduardo; Wang, Charles

2015-01-01

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. PMID:26657817

Fetal DNA does not induce preeclampsia-like symptoms when delivered in late pregnancy in the mouse.

PubMed

Čonka, Jozef; Konečná, Barbora; Lauková, Lucia; Vlková, Barbora; Celec, Peter

2017-04-01

The etiology of preeclampsia is unclear. Fetal DNA is present in higher concentrations in the plasma of pregnant women suffering from preeclampsia than in the plasma of healthy pregnant women. A previously published study has shown that human fetal DNA injected into pregnant mice induces preeclampsia-like symptoms when administered between gestation days 10-14. The aim of our experiment was to determine whether or not similar effects would be induced by administration of human and mouse fetal DNA, as well as mouse adult DNA and lipopolysaccharide during late pregnancy in the mouse. Experimental animals were injected daily intraperitoneally during gestation days 14-18 with either saline - negative control, lipopolysaccharide - positive control, or various types of DNA. On gestation day 19, blood pressure and proteinuria were measured, and placental and fetal weights were recorded. Fetal and placental hypotrophy were induced only by lipopolysaccharide (p < 0.001). Neither fetal nor adult DNA induced changes in fetal/placental weight. None of the experimental groups had higher blood pressure or urinary protein in comparison to saline treated animals. In our experiment, we found that there was no effect from intraperitoneally injected human fetal DNA, mouse fetal DNA, or mouse adult DNA on pregnant mice. Additionally, relatively high doses of various types of DNA did not induce preeclampsia-like symptoms in mice when administered in late pregnancy. Our negative results support the hypothesis that the increase of fetal DNA circulating in maternal circulation during the third trimester is rather a consequence than a cause of preeclampsia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea.

PubMed

Zhang, Zhi-Jian; Guan, Hong-Xia; Yang, Kun; Xiao, Bo-Kui; Liao, Hua; Jiang, Yang; Zhou, Tao; Hua, Qing-Quan

2017-10-01

This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL). Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells. Ouabain at the dosages of 0.5 mM, 1 mM and 3 mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3 mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure. The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.

Rod electrical coupling is controlled by a circadian clock and dopamine in mouse retina

PubMed Central

Jin, Nan Ge; Chuang, Alice Z; Masson, Philippe J; Ribelayga, Christophe P

2015-01-01

Key points Rod photoreceptors play a key role in vision in dim light; in the mammalian retina, although rods are anatomically connected or coupled by gap junctions, a type of electrical synapse, the functional importance and regulation of rod coupling has remained elusive. We have developed a new technique in the mouse: perforated patch-clamp recording of rod inner segments in isolated intact retinae maintained by superfusion. We find that rod electrical coupling is controlled by a circadian clock and dopamine, and is weak during the day and stronger at night. The results also indicate that the signal-to-noise ratio for a dim light response is increased at night because of coupling. Our observations will provide a framework for understanding the daily variations in human vision as well as the basis of specific retinal malfunctions. Abstract Rod single-photon responses are critical for vision in dim light. Electrical coupling via gap junction channels shapes the light response properties of vertebrate photoreceptors, but the regulation of rod coupling and its impact on the single-photon response have remained unclear. To directly address these questions, we developed a perforated patch-clamp recording technique and recorded from single rod inner segments in isolated intact neural mouse retinae, maintained by superfusion. Experiments were conducted at different times of the day or under constant environmental conditions, at different times across the circadian cycle. We show that rod electrical coupling is regulated by a circadian clock and dopamine, so that coupling is weak during the day and strong at night. Altogether, patch-clamp recordings of single-photon responses in mouse rods, tracer coupling, receptive field measurements and pharmacological manipulations of gap junction and dopamine receptor activity provide compelling evidence that rod coupling is modulated in a circadian manner. These data are consistent with computer modelling. At night, single

The in vitro mitogenic response to intact bacteria by murine B cells does not predict in vivo susceptibility to Salmonella typhimurium.

PubMed

Elkins, K; Metcalf, E S

1986-05-01

We are interested in developing in vitro culture systems that will permit immune responses to intact Salmonella typhimurium, since these systems would have certain advantages over in vivo infection models for the characterization of the host's responding cell types. In this report, the in vitro proliferative response of nonimmune murine spleen cells to four different killed preparations of S. typhimurium, strain TML (TML), are examined. These studies show that UV-killed TML, heat-killed TML, glutaraldehyde-killed TML, and acetone-killed and dried TML, all elicit a nonspecific mitogenic spleen cell response in vitro, as does a live, avirulent, temperature-sensitive mutant of TML, TS27. This response reaches a maximum on day 2 after initiation of culture, which is similar to the time course of a conventional lipopolysaccharide (LPS) response. Unlike the LPS response, little 3H-thymidine incorporation is observed in low-density cultures (2 X 10(5) cells/well), which suggests a critical role for accessory cells. The responding cell types include, but are not necessarily limited to, the B-cell population. The response cannot be readily inhibited by polymyxin B, which binds specifically to the lipid A portion of LPS. Thus, the bacterial components required for mitogenicity are not yet definitively identified. A survey of the mitogenic responses of lymphocytes from various inbred mouse strains, including the C3H/HeJ LPS hyporesponsive strain, indicates that all B cells tested are capable of proliferating vigorously in response to intact TML, regardless of the in vivo susceptibility to virulent infection. These results also emphasize the importance of assessing the nonspecific components of the immune response when studying the specific immune response to intact S. typhimurium.

Vulnerability of ecosystems to climate change moderated by habitat intactness.

PubMed

Eigenbrod, Felix; Gonzalez, Patrick; Dash, Jadunandan; Steyl, Ilse

2015-01-01

The combined effects of climate change and habitat loss represent a major threat to species and ecosystems around the world. Here, we analyse the vulnerability of ecosystems to climate change based on current levels of habitat intactness and vulnerability to biome shifts, using multiple measures of habitat intactness at two spatial scales. We show that the global extent of refugia depends highly on the definition of habitat intactness and spatial scale of the analysis of intactness. Globally, 28% of terrestrial vegetated area can be considered refugia if all natural vegetated land cover is considered. This, however, drops to 17% if only areas that are at least 50% wilderness at a scale of 48×48 km are considered and to 10% if only areas that are at least 50% wilderness at a scale of 4.8×4.8 km are considered. Our results suggest that, in regions where relatively large, intact wilderness areas remain (e.g. Africa, Australia, boreal regions, South America), conservation of the remaining large-scale refugia is the priority. In human-dominated landscapes, (e.g. most of Europe, much of North America and Southeast Asia), focusing on finer scale refugia is a priority because large-scale wilderness refugia simply no longer exist. Action to conserve such refugia is particularly urgent since only 1 to 2% of global terrestrial vegetated area is classified as refugia and at least 50% covered by the global protected area network. © 2014 John Wiley & Sons Ltd.

Proteomic characterization of histone variants in the mouse testis by mass spectrometry-based top-down analysis.

PubMed

Kwak, Ho-Geun; Dohmae, Naoshi

2016-11-15

Various histones, including testis-specific histones, exist during spermatogenesis and some of them have been reported to play a key role in chromatin remodeling. Mass spectrometry (MS)-based characterization has become the important step to understand histone structures. Although individual histones or partial histone variant groups have been characterized, the comprehensive analysis of histone variants has not yet been conducted in the mouse testis. Here, we present the comprehensive separation and characterization of histone variants from mouse testes by a top-down approach using MS. Histone variants were successfully separated on a reversed phase column using high performance liquid chromatography (HPLC) with an ion-pairing reagent. Increasing concentrations of testis-specific histones were observed in the mouse testis and some somatic histones increased in the epididymis. Specifically, the increase of mass abundance in H3.2 in the epididymis was inversely proportional to the decrease in H3t in the testis, which was approximately 80%. The top-down characterization of intact histone variants in the mouse testis was performed using LC-MS/MS. The masses of separated histone variants and their expected post-translation modifications were calculated by performing deconvolution with information taken from the database. TH2A, TH2B and H3t were characterized by MS/MS fragmentation. Our approach provides comprehensive knowledge for identification of histone variants in the mouse testis that will contribute to the structural and functional research of histone variants during spermatogenesis.

Mice with hepatocyte-specific deficiency of type 3 deiodinase have intact liver regeneration and accelerated recovery from nonthyroidal illness after toxin-induced hepatonecrosis.

PubMed

Castroneves, Luciana A; Jugo, Rebecca H; Maynard, Michelle A; Lee, Jennifer S; Wassner, Ari J; Dorfman, David; Bronson, Roderick T; Ukomadu, Chinweike; Agoston, Agoston T; Ding, Lai; Luongo, Cristina; Guo, Cuicui; Song, Huaidong; Demchev, Valeriy; Lee, Nicholas Y; Feldman, Henry A; Vella, Kristen R; Peake, Roy W; Hartigan, Christina; Kellogg, Mark D; Desai, Anal; Salvatore, Domenico; Dentice, Monica; Huang, Stephen A

2014-10-01

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.

Mice With Hepatocyte-Specific Deficiency of Type 3 Deiodinase Have Intact Liver Regeneration and Accelerated Recovery From Nonthyroidal Illness After Toxin-Induced Hepatonecrosis

PubMed Central

Castroneves, Luciana A.; Jugo, Rebecca H.; Maynard, Michelle A.; Lee, Jennifer S.; Wassner, Ari J.; Dorfman, David; Bronson, Roderick T.; Ukomadu, Chinweike; Agoston, Agoston T.; Ding, Lai; Luongo, Cristina; Guo, Cuicui; Song, Huaidong; Demchev, Valeriy; Lee, Nicholas Y.; Feldman, Henry A.; Vella, Kristen R.; Peake, Roy W.; Hartigan, Christina; Kellogg, Mark D.; Desai, Anal; Salvatore, Domenico; Dentice, Monica

2014-01-01

Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome. PMID:25004090

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult.

PubMed

Herrlinger, Stephanie A; Shao, Qiang; Ma, Li; Brindley, Melinda; Chen, Jian-Fu

2018-04-26

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

PubMed

Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

2016-01-01

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

PubMed

Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

2016-03-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. Copyright © 2016 Elsevier Ltd. All rights

BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

PubMed Central

Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

2016-01-01

Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

Xenopus egg cytoplasm with intact actin.

PubMed

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks.

PubMed

Valletta, Elisa; Kučera, Lukáš; Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks

PubMed Central

Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general. PMID:26821236

Isolation and culture of adult mouse vestibular nucleus neurons

PubMed

Him, Aydın; Altuntaş, Serap; Öztürk, Gürkan; Erdoğan, Ender; Cengiz, Nureddin

2017-12-19

Background/aim: Isolated cell cultures are widely used to study neuronal properties due to their advantages. Although embryonic animals are preferred for culturing, their morphological or electrophysiological properties may not reflect adult neurons, which may be important in neurodegenerative diseases. This paper aims to develop a method for preparing isolated cell cultures of medial vestibular nucleus (MVN) from adult mice and describe its morphological and electrophysiological properties.Materials and methods: Vestibular nucleus neurons were mechanically and enzymatically isolated and cultured using a defined medium with known growth factors. Cell survival was measured with propidium iodide, and electrophysiological properties were investigated with current-clamp recording.Results: Vestibular neurons grew neurites in cultures, gaining adult-like morphological properties, and stayed viable for 3 days in culture. Adding bovine calf serum, nerve growth factor, or insulin-like growth factor into the culture medium enhanced neuronal viability. Current-clamp recording of the cultured neurons revealed tonic and phasic-type neurons with similar input resistance, resting membrane potential, action potential amplitude, and duration. Conclusion: Vestibular neurons from adult mice can be cultured, and regenerate axons in a medium containing appropriate growth factors. Culturing adult vestibular neurons provides a new method to study age-related pathologies of the vestibular system.

Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

PubMed

Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

2014-09-09

Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Biodegradation of the ZnO:Eu nanoparticles in the tissues of adult mouse after alimentary application.

PubMed

Kielbik, Paula; Kaszewski, Jaroslaw; Rosowska, Julita; Wolska, Ewelina; Witkowski, Bartłomiej S; Gralak, Mikolaj A; Gajewski, Zdzisław; Godlewski, Marek; Godlewski, Michal M

2017-04-01

Biodegradable zinc oxide nanoparticles (ZnO NPs) are considered promising materials for future biomedical applications. To fulfil this potential, biodistribution and elimination patterns of ZnO NPs in the living organism need to be resolved. In order to investigate gastrointestinal absorption of ZnO NPs and their intra-organism distribution, water suspension of ZnO or fluorescent ZnO:Eu (Europium-doped zinc oxide) NPs (10mg/ml; 0.3ml/mouse) was alimentary-administered (IG: intra-gastric) to adult mice. Internal organs collected at key time-points after IG were evaluated by AAS for Zn concentration and analysed by cytometric techniques. We found that Zn-based NPs were readily absorbed and distributed (3 h post IG) in the nanoparticle form throughout the organism. Results suggest, that liver and kidneys were key organs responsible for NPs elimination, while accumulation was observed in the spleen and adipose tissues. We also showed that ZnO/ZnO:Eu NPs were able to cross majority of biological barriers in the organism (including blood-brain-barrier). Copyright © 2016 Elsevier Inc. All rights reserved.

Germline competency of parthenogenetic embryonic stem cells from immature oocytes of adult mouse ovary

PubMed Central

Liu, Zhong; Hu, Zhe; Pan, Xinghua; Li, Minshu; Togun, Taiwo A.; Tuck, David; Pelizzola, Mattia; Huang, Junjiu; Ye, Xiaoying; Yin, Yu; Liu, Mengyuan; Li, Chao; Chen, Zhisheng; Wang, Fang; Zhou, Lingjun; Chen, Lingyi; Keefe, David L.; Liu, Lin

2011-01-01

Parthenogenetic embryonic stem cells (pESCs) have been generated in several mammalian species from parthenogenetic embryos that would otherwise die around mid-gestation. However, previous reports suggest that pESCs derived from in vivo ovulated (IVO) mature oocytes show limited pluripotency, as evidenced by low chimera production, high tissue preference and especially deficiency in germline competence, a critical test for genetic integrity and pluripotency of ESCs. Here, we report efficient generation of germline-competent pESC lines (named as IVM pESCs) from parthenogenetic embryos developed from immature oocytes of adult mouse ovaries following in vitro maturation (IVM) and artificial activation. In contrast, pESCs derived from IVO oocytes show defective germline competence, consistent with previous reports. Further, IVM pESCs resemble more ESCs from fertilized embryos (fESCs) than do IVO pESCs on genome-wide DNA methylation and global protein profiles. In addition, IVM pESCs express higher levels of Blimp1, Lin28 and Stella, relative to fESCs, and in their embryoid bodies following differentiation. This may indicate differences in differentiation potentially to the germline. The mechanisms for acquisition of pluripotency and germline competency of IVM pESCs from immature oocytes remain to be determined. PMID:21239471

Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

PubMed

Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

2017-09-01

Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

A Comparison of Bacterial Composition in Diabetic Ulcers and Contralateral Intact Skin

PubMed Central

Gontcharova, Viktoria; Youn, Eunseog; Sun, Yan; Wolcott, Randall D; Dowd, Scot E

2010-01-01

An extensive portion of the healthcare budget is allocated to chronic human infection. Chronic wounds in particular are a major contributor to this financial burden. Little is known about the types of bacteria which may contribute to the chronicity, biofilm and overall bioburden of the wound itself. In this study we compare the bacteriology of wounds and associated intact skin. Wound and paired intact skin swabs (from a contralateral location) were collected. The bacterial diversity was determined using bacterial Tag-encoded FLX amplicon pyrosequencing (bTEFAP). Diversity analysis showed intact skin to be significantly more diverse than wounds on both the species and genus levels (3% and 5% divergence). Furthermore, wounds show heightened levels of anaerobic bacteria, like Peptoniphilus, Finegoldia, and Anaerococcus, and other detrimental genera such as Corynebacterium and Staphylococcus. Although some of these and other bacterial genera were found to be common between intact skin and wounds, notable opportunistic wound pathogens were found at lower levels in intact skin. Principal Component Analysis demonstrated a clear separability of the two groups. The findings of the study not only greatly support the hypothesis of differing bacterial composition of intact skin and wounds, but also contribute additional insight into the ecology of skin and wound microflora. The increased diversity and lowered levels of opportunistic pathogens found in skin make the system highly distinguishable from wounds. PMID:20461221

Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient

PubMed Central

Klyen, Blake R.; Scolaro, Loretta; Shavlakadze, Tea; Grounds, Miranda D.; Sampson, David D.

2014-01-01

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient µt using optical coherence tomography. The resulting values of the local total attenuation coefficient µt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm−1) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm−1), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm−1) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm−1) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy. PMID:24761302

Modified madigan prostatectomy: a procedure preserved prostatic urethra intact.

PubMed

Lu, Jun; Ye, Zhangqun; Hu, Weilie

2005-01-01

A total of 92 patients with benign prostatic hyperplasia (BPH) were subjected to modified Madigan prostatectomy (MPC) for a much satisfactory effect in open prostatectomy surgery. Exposing anterior prostatic urethra near the bladder neck and conjunct cystotomy modified the MPC procedure. This modified procedure preserved prostatic urethra intact and could also deal with intracystic lesions at the same time. The intact of prostatic urethra was kept completely or largely in 86 cases. The amount of blood loss during modified procedure was less. The mean operative time was 105 min. Seventy patients had been followed up for 3-24 months. The postoperative average Qmax was 19. 2 ml/s. The cystourethrography revealed that the urethra and bladder neck were intact in 10 patients postoperatively. Furthermore, the prostatic urethra was obviously wider after modified MPC. The modified MPC can reduce the occurrence of urethra injury and enlarge the MPC indications. The modified technique is easy to perform with less complications and much satisfactory clinical result.

Arraying of intact liposomes into chemically functionalized microwells.

PubMed

Kalyankar, Nikhil D; Sharma, Manoj K; Vaidya, Shyam V; Calhoun, David; Maldarelli, Charles; Couzis, Alexander; Gilchrist, Lane

2006-06-06

Here, we describe a protocol to bind individual, intact phospholipid bilayer liposomes, which are on the order of 1 microm in diameter, in microwells etched in a regular array on a silicon oxide substrate. The diameter of the wells is on the order of the liposome diameter, so only one liposome is located in each well. The background of the silicon oxide surface is functionalized with a PEG oligomer using the contact printing of a PEG silane to present a surface that resists the adsorption of proteins, lipid material, and liposomes. The interiors of the wells are functionalized with an aminosilane to facilitate the conjugation of biotin, which is then bound to Neutravidin. The avidin-coated well interiors bind the liposomes whose surfaces contain biotinylated lipids. The specific binding of the liposomes to the surface using the biotin-avidin linkage, together with the resistant nature of the background and the physical confinement of the wells, allows the liposomes to remain intact and to not unravel, rupture, and fuse onto the surface. We demonstrate this intact arraying using confocal laser scanning microscopy of fluorophores specifically tagging the microwells, the lipid bilayer, and the aqueous interior of the liposome.

Hemodynamic and morphologic responses in mouse brain during acute head injury imaged by multispectral structured illumination

NASA Astrophysics Data System (ADS)

Volkov, Boris; Mathews, Marlon S.; Abookasis, David

2015-03-01

Multispectral imaging has received significant attention over the last decade as it integrates spectroscopy, imaging, tomography analysis concurrently to acquire both spatial and spectral information from biological tissue. In the present study, a multispectral setup based on projection of structured illumination at several near-infrared wavelengths and at different spatial frequencies is applied to quantitatively assess brain function before, during, and after the onset of traumatic brain injury in an intact mouse brain (n=5). For the production of head injury, we used the weight drop method where weight of a cylindrical metallic rod falling along a metal tube strikes the mouse's head. Structured light was projected onto the scalp surface and diffuse reflected light was recorded by a CCD camera positioned perpendicular to the mouse head. Following data analysis, we were able to concurrently show a series of hemodynamic and morphologic changes over time including higher deoxyhemoglobin, reduction in oxygen saturation, cell swelling, etc., in comparison with baseline measurements. Overall, results demonstrates the capability of multispectral imaging based structured illumination to detect and map of brain tissue optical and physiological properties following brain injury in a simple noninvasive and noncontact manner.

Implicit Learning Deficits among Adults with Developmental Dyslexia

ERIC Educational Resources Information Center

Kahta, Shani; Schiff, Rachel

2016-01-01

The aim of the present study was to investigate implicit learning processes among adults with developmental dyslexia (DD) using a visual linguistic artificial grammar learning (AGL) task. Specifically, it was designed to explore whether the intact learning reported in previous studies would also occur under conditions including minimal training…

Gist-based conceptual processing of pictures remains intact in patients with amnestic mild cognitive impairment.

PubMed

Deason, Rebecca G; Hussey, Erin P; Budson, Andrew E; Ally, Brandon A

2012-03-01

The picture superiority effect, better memory for pictures compared to words, has been found in young adults, healthy older adults, and, most recently, in patients with Alzheimer's disease and mild cognitive impairment. Although the picture superiority effect is widely found, there is still debate over what drives this effect. One main question is whether it is enhanced perceptual or conceptual information that leads to the advantage for pictures over words. In this experiment, we examined the picture superiority effect in healthy older adults and patients with amnestic mild cognitive impairment (MCI) to better understand the role of gist-based conceptual processing. We had participants study three exemplars of categories as either words or pictures. In the test phase, participants were again shown pictures or words and were asked to determine whether the item was in the same category as something they had studied earlier or whether it was from a new category. We found that all participants demonstrated a robust picture superiority effect, better performance for pictures than for words. These results suggest that the gist-based conceptual processing of pictures is preserved in patients with MCI. While in healthy older adults preserved recollection for pictures could lead to the picture superiority effect, in patients with MCI it is most likely that the picture superiority effect is a result of spared conceptually based familiarity for pictures, perhaps combined with their intact ability to extract and use gist information.

Gist-based conceptual processing of pictures remains intact in patients with amnestic mild cognitive impairment

PubMed Central

Deason, Rebecca G.; Hussey, Erin P.; Budson, Andrew E.; Ally, Brandon A.

2012-01-01

Objective The picture superiority effect, better memory for pictures compared to words, has been found in young adults, healthy older adults, and, most recently, in patients with Alzheimer’s disease and mild cognitive impairment. Although the picture superiority effect is widely found, there is still debate over what drives this effect. One main question is whether it is enhanced perceptual or conceptual information that leads to the advantage for pictures over words. In this experiment, we examined the picture superiority effect in healthy older adults and patients with amnestic mild cognitive impairment (MCI) to better understand the role of gist-based conceptual processing. Method We had participants study three exemplars of categories as either words or pictures. In the test phase, participants were again shown pictures or words and were asked to determine whether the item was in the same category as something they had studied earlier or whether it was from a new category. Results We found that all participants demonstrated a robust picture superiority effect, better performance for pictures than for words. Conclusions These results suggest that the gist-based conceptual processing of pictures is preserved in patients with MCI. While in healthy older adults preserved recollection for pictures could lead to the picture superiority effect, in patients with MCI it is most likely that the picture superiority effect is a result of spared conceptually-based familiarity for pictures, perhaps combined with their intact ability to extract and use gist information. PMID:22229341

The Influence of Negative Emotion on Cognitive and Emotional Control Remains Intact in Aging

PubMed Central

Zinchenko, Artyom; Obermeier, Christian; Kanske, Philipp; Schröger, Erich; Villringer, Arno; Kotz, Sonja A.

2017-01-01

Healthy aging is characterized by a gradual decline in cognitive control and inhibition of interferences, while emotional control is either preserved or facilitated. Emotional control regulates the processing of emotional conflicts such as in irony in speech, and cognitive control resolves conflict between non-affective tendencies. While negative emotion can trigger control processes and speed up resolution of both cognitive and emotional conflicts, we know little about how aging affects the interaction of emotion and control. In two EEG experiments, we compared the influence of negative emotion on cognitive and emotional conflict processing in groups of younger adults (mean age = 25.2 years) and older adults (69.4 years). Participants viewed short video clips and either categorized spoken vowels (cognitive conflict) or their emotional valence (emotional conflict), while the visual facial information was congruent or incongruent. Results show that negative emotion modulates both cognitive and emotional conflict processing in younger and older adults as indicated in reduced response times and/or enhanced event-related potentials (ERPs). In emotional conflict processing, we observed a valence-specific N100 ERP component in both age groups. In cognitive conflict processing, we observed an interaction of emotion by congruence in the N100 responses in both age groups, and a main effect of congruence in the P200 and N200. Thus, the influence of emotion on conflict processing remains intact in aging, despite a marked decline in cognitive control. Older adults may prioritize emotional wellbeing and preserve the role of emotion in cognitive and emotional control. PMID:29163132

The Influence of Negative Emotion on Cognitive and Emotional Control Remains Intact in Aging.

PubMed

Zinchenko, Artyom; Obermeier, Christian; Kanske, Philipp; Schröger, Erich; Villringer, Arno; Kotz, Sonja A

2017-01-01

Healthy aging is characterized by a gradual decline in cognitive control and inhibition of interferences, while emotional control is either preserved or facilitated. Emotional control regulates the processing of emotional conflicts such as in irony in speech, and cognitive control resolves conflict between non-affective tendencies. While negative emotion can trigger control processes and speed up resolution of both cognitive and emotional conflicts, we know little about how aging affects the interaction of emotion and control. In two EEG experiments, we compared the influence of negative emotion on cognitive and emotional conflict processing in groups of younger adults (mean age = 25.2 years) and older adults (69.4 years). Participants viewed short video clips and either categorized spoken vowels (cognitive conflict) or their emotional valence (emotional conflict), while the visual facial information was congruent or incongruent. Results show that negative emotion modulates both cognitive and emotional conflict processing in younger and older adults as indicated in reduced response times and/or enhanced event-related potentials (ERPs). In emotional conflict processing, we observed a valence-specific N100 ERP component in both age groups. In cognitive conflict processing, we observed an interaction of emotion by congruence in the N100 responses in both age groups, and a main effect of congruence in the P200 and N200. Thus, the influence of emotion on conflict processing remains intact in aging, despite a marked decline in cognitive control. Older adults may prioritize emotional wellbeing and preserve the role of emotion in cognitive and emotional control.

Tidal stretches do not modulate responsiveness of intact airways in vitro

PubMed Central

Szabo, Thomas L.; Suki, Béla; Lutchen, Kenneth R.

2010-01-01

Studies on isolated tracheal airway smooth muscle (ASM) strips have shown that length/force fluctuations, similar to those likely occurring during breathing, will mitigate ASM contractility. These studies conjecture that, solely by reducing length oscillations on a healthy, intact airway, one can create airway hyperresponsiveness, but this has never been explicitly tested. The intact airway has additional complexities of geometry and structure that may impact its relevance to isolated ASM strips. We examined the role of transmural pressure (Ptm) fluctuations of physiological amplitudes on the responsiveness of an intact airway. We developed an integrated system utilizing ultrasound imaging to provide real-time measurements of luminal radius and wall thickness over the full length of an intact airway (generation 10 and below) during Ptm oscillations. First, airway constriction dynamics to cumulative acetylcholine (ACh) doses (10−7 to 10−3 M) were measured during static and dynamic Ptm protocols. Regardless of the breathing pattern, the Ptm oscillation protocols were ineffective in reducing the net level of constriction for any ACh dose, compared with the static control (P = 0.225–0.793). Next, Ptm oscillations of increasing peak-to-peak amplitude were applied subsequent to constricting intact airways under static conditions (5.0-cmH2O Ptm) with a moderate ACh dose (10−5 M). Peak-to-peak Ptm oscillations ≤5.0 cmH2O resulted in no statistically significant bronchodilatory response (P = 0.429 and 0.490). Larger oscillations (10 cmH2O, peak to peak) produced modest dilation of 4.3% (P = 0.009). The lack of modulation of airway responsiveness by Ptm oscillations in intact, healthy airways suggests that ASM level mechanisms alone may not be the sole determinant of airway responsiveness. PMID:20431023

Time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain.

PubMed

Khlghatyan, Jivan; Saghatelyan, Armen

2012-09-12

the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

Memory deficits with intact cognitive control in the methylazoxymethanol acetate (MAM) exposure model of neurodevelopmental insult.

PubMed

O'Reilly, Kally C; Perica, Maria I; Fenton, André A

2016-10-01

Cognitive impairments are amongst the most debilitating deficits of schizophrenia and the best predictor of functional outcome. Schizophrenia is hypothesized to have a neurodevelopmental origin, making animal models of neurodevelopmental insult important for testing predictions that early insults will impair cognitive function. Rats exposed to methylazoxymethanol acetate (MAM) at gestational day 17 display morphological, physiological and behavioral abnormalities relevant to schizophrenia. Here we investigate the cognitive abilities of adult MAM rats. We examined brain activity in MAM rats by histochemically assessing cytochrome oxidase enzyme activity, a metabolic marker of neuronal activity. To assess cognition, we used a hippocampus-dependent two-frame active place avoidance paradigm to examine learning and spatial memory, as well as cognitive control and flexibility using the same environment and evaluating the same set of behaviors. We confirmed that adult MAM rats have altered hippocampal morphology and brain function, and that they are hyperactive in an open field. The latter likely indicates MAM rats have a sensorimotor gating deficit that is common to many animal models used for schizophrenia research. On first inspection, cognitive control seems impaired in MAM rats, indicated by more errors during the two-frame active place avoidance task. Because MAM rats are hyperactive throughout place avoidance training, we considered the possibility that the hyperlocomotion may account for the apparent cognitive deficits. These deficits were reduced on the basis of measures of cognitive performance that account for motor activity differences. However, though other aspects of memory are intact, the ability of MAM rats to express trial-to-trial memory is delayed compared to control rats. These findings suggest that spatial learning and cognitive abilities are largely intact, that the most prominent cognitive deficit is specific to acquiring memory in the MAM

Mechanism of autoregulation in the intact kidney.

DOT National Transportation Integrated Search

1963-12-01

The mechanism of renal autoregulation is unclear. The subject of the present investigation is the mechanism of autoregulation of blood flow in the intact kidney. The left kidney of the anesthetized dog was exposed via a flank approach. Renal venous o...

Self-etching adhesive on intact enamel, with and without pre-etching.

PubMed

Devarasa, G M; Subba Reddy, V V; Chaitra, N L; Swarna, Y M

2012-05-01

Bond strengths of composite resin to enamel using self-etch adhesive (SEA) Clearfil SE bond system on intact enamel and enamel pre-etched with phosphoric acid were compared. The objective was to determine if the pre-etching would increase the bond strengths of the SEA systems to intact enamel and to evaluate the effect of pre-etching on bond formation of self-etch adhesives on intact enamel. Labial surfaces of 40 caries free permanent upper central and lateral incisors were cleaned, sectioned of their roots. All specimens were mounted on acrylic block and divided randomly into four groups. In two groups the application of self-etch adhesive, Clearfil SE bond was carried as per manufacturer's instructions, composite cylinders were built, whereas in the other two groups, 37% phosphoric acid etching was done before the application of self-etching adhesives. Then the resin tags were analyzed using scanning electron microscope and shear bond strength was measured using Instron universal testing machine. When phosphoric acid was used, there was significant increase in the depth of penetration of resin tags and in the Shear Bond Strength of composite to enamel. The results indicate that out of both treatment groups, pre-etching the intact enamel with 37% phosphoric acid resulted in formation of longer resin tags and higher depth of penetration of resin tags of the Clearfil SE bond, and attaining higher bond strength of the Clearfil SE bond to intact enamel. Copyright © 2011 Wiley Periodicals, Inc.

Threats to intact tropical peatlands and opportunities for their conservation.

PubMed

Roucoux, K H; Lawson, I T; Baker, T R; Del Castillo Torres, D; Draper, F C; Lähteenoja, O; Gilmore, M P; Honorio Coronado, E N; Kelly, T J; Mitchard, E T A; Vriesendorp, C F

2017-12-01

Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza-Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land-use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon-dense (domed pole forest) areas. New carbon-based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development. © 2017 The Authors. Conservation Biology published by Wiley Periodicals, Inc

Brief Report: The Role of Task Support in the Spatial and Temporal Source Memory of Adults with Autism Spectrum Disorder

ERIC Educational Resources Information Center

Bowler, Dermot M.; Gaigg, Sebastian B.; Gardiner, John M.

2015-01-01

Adults with autism spectrum disorder (ASD) show intact recognition (supported procedure) but impaired recall (unsupported procedure) of incidentally-encoded context. Because this has not been demonstrated for temporal source, we compared the temporal and spatial source memory of adults with ASD and verbally matched typical adults. Because of…

HYDROCARBON VAPOR DIFFUSION IN INTACT CORE SLEEVES

EPA Science Inventory

The diffusion of 2,2,4-trimethylpentane (TMP) and 2,2,5-trimethylhexane (TMH) vapors put of residually contaminated sandy soil from the U.S. Environmental Protection Agency (EPA) field research site at Traverse City, Michigan, was measured and modeled. The headspace of an intact ...

The use of injection-corrosive method in the study of extraorganic bloodstream of human intact stomach.

PubMed

Hryn, V H; Svintsytska, N L; Piliuhin, V; Ustenko, R L; Katsenko, A L

Functional and morphological state of the organs and tissues mainly depends on the adequate blood supply and lymph movement, function of which is integrated by the nervous system. A crucial link in the morphogenesis of the gastric lesions is the intensity of vascularization, as well as the fact that in its venous part the gastric bloodstream is almost entirely included into the portal vein system. Knowledge of the anatomy of the normal human stomach conditions is of indispensable practical value, since they are required for the proper interpretation of the pathological changes occurred in it. To obtain the spatial visual information about the angioarchitecture of the extraorganic bloodstream of human intact stomach deep in the gastric wall. 10 post-autopsy adult total stomach specimens of patients, died for the reasons not associated with manifested gastrointestinal diseases have been analyzed. The specimens were extracted during the dissection together with portions of lesser and greater omentum, and segment of aorta with celiac trunk. To neutralize the acidic contents of the stomach, its cavity was washed by 4% sodium bicarbonate solution with subsequent wash in warm running water. The vascular injection method with subsequent corrosion of soft tissues was used in investigation of gastric bloodstream. On the basis of the investigations the advantages of the countercurrent-crossing method of injection of extraorganic vessels to fill the bloodstream of human stomach have been discussed. Positive results of the suggested technique for morphological study of blood vessels have been noted. The three-dimensional spatial organization of the extraorganic bloodstream of the intact stomach can be studied on the basis of the injection-corrosive casts. Thus, the use of the suggested method enables to obtain the fine three-dimensional reproduction of extraorganic bloodstream of the human stomach. The obtained high-quality casts, in turn, are used for the subsequent

Development of a novel mouse glioma model using lentiviral vectors

PubMed Central

Marumoto, Tomotoshi; Tashiro, Ayumu; Friedmann-Morvinski, Dinorah; Scadeng, Miriam; Soda, Yasushi; Gage, Fred H; Verma, Inder M

2009-01-01

We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice. PMID:19122659

Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18.

PubMed

Hu, Biliang; Ren, Jiangtao; Luo, Yanping; Keith, Brian; Young, Regina M; Scholler, John; Zhao, Yangbing; June, Carl H

2017-09-26

The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR) T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE) CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors. Copyright © 2017. Published by Elsevier Inc.

Bioavailability in healthy adults of efavirenz capsule contents mixed with a small amount of food.

PubMed

Kaul, Sanjeev; Ji, Ping; Lu, Michael; Nguyen, Kim L; Shangguan, Tong; Grasela, Dennis

2010-02-01

The effect of mixing the contents of efavirenz capsules (sprinkles) with a small amount of food on the bioavailability and pharmacokinetics of efavirenz in healthy adults was evaluated. In a randomized, three-period, crossover study, 24 healthy adult subjects were divided equally into two groups. Group I received treatments A, B, and C, and those in group II received treatments A, D, and E. Treatment A was three efavirenz 200-mg intact capsules under fasting conditions. Treatments B, C, D, and E were three efavirenz 200-mg capsule contents mixed with two teaspoons of applesauce, grape jelly, yogurt, or infant formula, respectively. A single dose was given on days 1, 21, and 41. The steady-state mean maximum observed concentration, time of maximum observed concentration, area under the concentration-time curve (AUC) half-life, taste, and safety were assessed. The AUC after administration of a single 600-mg dose of efavirenz sprinkles mixed with two teaspoons of any of the food vehicles to healthy adults was bioequivalent to a 600-mg efavirenz dose given as intact capsules under fasting conditions. Subjects rated efavirenz mixed with grape jelly as the most palatable. Adverse events and laboratory abnormalities were similar for all treatments. The AUC of efavirenz 600 mg administered as capsule sprinkles with two teaspoons of applesauce, grape jelly, yogurt, or infant formula was bioequivalent to a single dose of efavirenz 600 mg given as intact capsules under fasting conditions in healthy adults.

Source Selection Simulation: Intact Team Training on Picking a Provider

DTIC Science & Technology

2015-06-01

seat of a new $100 million stealth fighter before giving her flight simulation time. The ar- gument for source-selection simulation ( SSS ) training is...dynamic is the creation of the SSS Tool. Drawing on his success in using a similar tool in contingency contracting, Long decided we should use a Web...of SSS intact team training. On Sept. 30–Oct. 3, 2014, Professors Long and Elsesser de- livered DAU’s first-ever Intact Team SSS Training to Eglin’s

Tracking Intact Phospholipids and Triacylglycerides in Bering Sea Euphausiids during Two Pulsed Feeding Experiments via Tandem LC-MS

NASA Astrophysics Data System (ADS)

Pleuthner, R. L.; Harvey, H. R.

2016-02-01

In the eastern Bering Sea and Chukchi Sea, Thysanoessa raschii are the most abundant krill species and a keystone trophic component that serves as both an important grazer and link to upper levels consumers including whales. Krill experience large variation in food resources annually and store multiple lipid classes for both reproduction and growth. Two shipboard feeding experiments tested the lipid retention in adult T. raschii and examined the fluctuation of specific lipid biomarkers under food-limited conditions. Phospholipids represent the major structural and storage lipids; their retention as intact phospholipids (IPL), as well as other glycerides (i.e. diacyl- and triacylglycerides; DG and TG), were followed over 19- and 31-day experiments using RPLC ESI-MS/MS on an LTQ Orbitrap XL. Identification and quantification of the suite of phospholipids and associated fatty acids with each experiment was performed with Lipid Search software. IPL's comprised the majority of intact lipids present, most of which had phosphatidylcholine (PC) headgroups; smaller contributions were made by phosphatidylethanolamine (PE) and phosphatidylserine (PS)-contaning IPL's. Fatty acids were largely represented by seven compounds - C14:0n, C16:0n, C16:1(n-7), C18:1(n-7), C18:1(n-9), C20:5(n-3), C22:6(n-3) - and were typically present as mixed acyl groups within each intact lipid class. Concentrations (μmole/g wet weight) of IPL and glyceride lipids showed a decrease of 21% and 26%, respectively, from initial values, suggesting that both are mobilized in times of food scarcity and during overwintering. Structures containing 16:1 decreased most for IPL's, reflecting the absence of the 16:1(n-7) dietary algal fatty acid. This powerful set of analytical and software tools allows determination of the suite of intact lipids within euphausiids to provide a more comprehensive picture of krill structural and storage lipids and their retention during times of varied food availability.

The longest telomeres: a general signature of adult stem cell compartments

PubMed Central

Flores, Ignacio; Canela, Andres; Vera, Elsa; Tejera, Agueda; Cotsarelis, George; Blasco, María A.

2008-01-01

Identification of adult stem cells and their location (niches) is of great relevance for regenerative medicine. However, stem cell niches are still poorly defined in most adult tissues. Here, we show that the longest telomeres are a general feature of adult stem cell compartments. Using confocal telomere quantitative fluorescence in situ hybridization (telomapping), we find gradients of telomere length within tissues, with the longest telomeres mapping to the known stem cell compartments. In mouse hair follicles, we show that cells with the longest telomeres map to the known stem cell compartments, colocalize with stem cell markers, and behave as stem cells upon treatment with mitogenic stimuli. Using K15-EGFP reporter mice, which mark hair follicle stem cells, we show that GFP-positive cells have the longest telomeres. The stem cell compartments in small intestine, testis, cornea, and brain of the mouse are also enriched in cells with the longest telomeres. This constitutes the description of a novel general property of adult stem cell compartments. Finally, we make the novel finding that telomeres shorten with age in different mouse stem cell compartments, which parallels a decline in stem cell functionality, suggesting that telomere loss may contribute to stem cell dysfunction with age. PMID:18283121

Preconditioning by isoflurane elicits mitochondrial protective mechanisms independent of sarcolemmal KATP channel in mouse cardiomyocytes

PubMed Central

Muravyeva, Maria; Sedlic, Filip; Dolan, Nicholas; Bosnjak, Zeljko J; Stadnicka, Anna

2013-01-01

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning (APC). Changes in mitochondrial bioenergetics influence the sarcKATP channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial ROS production and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180±14% and 190±15% and ROS production to 118±2% and 124±6% of baseline in WT and Kir6.2 KO myocytes, respectively. TMRE fluorescence decreased to 84±6% in WT and to 86±4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31±1% to 21±1% in WT and from 44±2% to 35±2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases sensitivity of intact cardiomyocytes t o oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in APC by isoflurane. PMID:23318991

46 CFR 174.045 - Intact stability requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045...

46 CFR 174.045 - Intact stability requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045...

Inducible and Conditional Deletion of Extracellular Signal-regulated Kinase 5 Disrupts Adult Hippocampal Neurogenesis*

PubMed Central

Pan, Yung-Wei; Zou, Junhui; Wang, Wenbin; Sakagami, Hiroyuki; Garelick, Michael G.; Abel, Glen; Kuo, Chay T.; Storm, Daniel R.; Xia, Zhengui

2012-01-01

Recent studies have led to the exciting idea that adult-born neurons in the dentate gyrus of the hippocampus may play a role in hippocampus-dependent memory formation. However, signaling mechanisms that regulate adult hippocampal neurogenesis are not well defined. Here we report that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, is selectively expressed in the neurogenic regions of the adult mouse brain. We present evidence that shRNA suppression of ERK5 in adult hippocampal neural stem/progenitor cells (aNPCs) reduces the number of neurons while increasing the number of cells expressing markers for stem/progenitor cells or proliferation. Furthermore, shERK5 attenuates both transcription and neuronal differentiation mediated by Neurogenin 2, a transcription factor expressed in adult hippocampal neural progenitor cells. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, promotes neurogenesis in cultured aNPCs and in the dentate gyrus of the mouse brain. Moreover, neurotrophins including NT3 activate ERK5 and stimulate neuronal differentiation in aNPCs in an ERK5-dependent manner. Finally, inducible and conditional deletion of ERK5 specifically in the neurogenic regions of the adult mouse brain delays the normal progression of neuronal differentiation and attenuates adult neurogenesis in vivo. These data suggest ERK5 signaling as a critical regulator of adult hippocampal neurogenesis. PMID:22645146

Visual speech alters the discrimination and identification of non-intact auditory speech in children with hearing loss.

PubMed

Jerger, Susan; Damian, Markus F; McAlpine, Rachel P; Abdi, Hervé

2017-03-01

Understanding spoken language is an audiovisual event that depends critically on the ability to discriminate and identify phonemes yet we have little evidence about the role of early auditory experience and visual speech on the development of these fundamental perceptual skills. Objectives of this research were to determine 1) how visual speech influences phoneme discrimination and identification; 2) whether visual speech influences these two processes in a like manner, such that discrimination predicts identification; and 3) how the degree of hearing loss affects this relationship. Such evidence is crucial for developing effective intervention strategies to mitigate the effects of hearing loss on language development. Participants were 58 children with early-onset sensorineural hearing loss (CHL, 53% girls, M = 9;4 yrs) and 58 children with normal hearing (CNH, 53% girls, M = 9;4 yrs). Test items were consonant-vowel (CV) syllables and nonwords with intact visual speech coupled to non-intact auditory speech (excised onsets) as, for example, an intact consonant/rhyme in the visual track (Baa or Baz) coupled to non-intact onset/rhyme in the auditory track (/-B/aa or/-B/az). The items started with an easy-to-speechread/B/or difficult-to-speechread/G/onset and were presented in the auditory (static face) vs. audiovisual (dynamic face) modes. We assessed discrimination for intact vs. non-intact different pairs (e.g., Baa:/-B/aa). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more same-as opposed to different-responses in the audiovisual than auditory mode. We assessed identification by repetition of nonwords with non-intact onsets (e.g.,/-B/az). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more Baz-as opposed to az- responses in the audiovisual than auditory mode. Performance in the audiovisual mode showed more same

Visual Speech Alters the Discrimination and Identification of Non-Intact Auditory Speech in Children with Hearing Loss

PubMed Central

Jerger, Susan; Damian, Markus F.; McAlpine, Rachel P.; Abdi, Hervé

2017-01-01

Objectives Understanding spoken language is an audiovisual event that depends critically on the ability to discriminate and identify phonemes yet we have little evidence about the role of early auditory experience and visual speech on the development of these fundamental perceptual skills. Objectives of this research were to determine 1) how visual speech influences phoneme discrimination and identification; 2) whether visual speech influences these two processes in a like manner, such that discrimination predicts identification; and 3) how the degree of hearing loss affects this relationship. Such evidence is crucial for developing effective intervention strategies to mitigate the effects of hearing loss on language development. Methods Participants were 58 children with early-onset sensorineural hearing loss (CHL, 53% girls, M = 9;4 yrs) and 58 children with normal hearing (CNH, 53% girls, M = 9;4 yrs). Test items were consonant-vowel (CV) syllables and nonwords with intact visual speech coupled to non-intact auditory speech (excised onsets) as, for example, an intact consonant/rhyme in the visual track (Baa or Baz) coupled to non-intact onset/rhyme in the auditory track (/–B/aa or /–B/az). The items started with an easy-to-speechread /B/ or difficult-to-speechread /G/ onset and were presented in the auditory (static face) vs. audiovisual (dynamic face) modes. We assessed discrimination for intact vs. non-intact different pairs (e.g., Baa:/–B/aa). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more same—as opposed to different—responses in the audiovisual than auditory mode. We assessed identification by repetition of nonwords with non-intact onsets (e.g., /–B/az). We predicted that visual speech would cause the non-intact onset to be perceived as intact and would therefore generate more Baz—as opposed to az— responses in the audiovisual than auditory mode. Results

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

PubMed

Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

2012-07-01

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

Tissue distribution and developmental expression of type XVI collagen in the mouse.

PubMed

Lai, C H; Chu, M L

1996-04-01

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

The familial dysautonomia disease gene IKBKAP is required in the developing and adult mouse central nervous system

PubMed Central

Chaverra, Marta; George, Lynn; Thorne, Julian; Grindeland, Andrea; Ueki, Yumi; Eiger, Steven; Cusick, Cassie; Babcock, A. Michael; Carlson, George A.

2017-01-01

ABSTRACT Hereditary sensory and autonomic neuropathies (HSANs) are a genetically and clinically diverse group of disorders defined by peripheral nervous system (PNS) dysfunction. HSAN type III, known as familial dysautonomia (FD), results from a single base mutation in the gene IKBKAP that encodes a scaffolding unit (ELP1) for a multi-subunit complex known as Elongator. Since mutations in other Elongator subunits (ELP2 to ELP4) are associated with central nervous system (CNS) disorders, the goal of this study was to investigate a potential requirement for Ikbkap in the CNS of mice. The sensory and autonomic pathophysiology of FD is fatal, with the majority of patients dying by age 40. While signs and pathology of FD have been noted in the CNS, the clinical and research focus has been on the sensory and autonomic dysfunction, and no genetic model studies have investigated the requirement for Ikbkap in the CNS. Here, we report, using a novel mouse line in which Ikbkap is deleted solely in the nervous system, that not only is Ikbkap widely expressed in the embryonic and adult CNS, but its deletion perturbs both the development of cortical neurons and their survival in adulthood. Primary cilia in embryonic cortical apical progenitors and motile cilia in adult ependymal cells are reduced in number and disorganized. Furthermore, we report that, in the adult CNS, both autonomic and non-autonomic neuronal populations require Ikbkap for survival, including spinal motor and cortical neurons. In addition, the mice developed kyphoscoliosis, an FD hallmark, indicating its neuropathic etiology. Ultimately, these perturbations manifest in a developmental and progressive neurodegenerative condition that includes impairments in learning and memory. Collectively, these data reveal an essential function for Ikbkap that extends beyond the peripheral nervous system to CNS development and function. With the identification of discrete CNS cell types and structures that depend on Ikbkap

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL).

PubMed

Kwon, Young Min; Chung, Hee Sun; Moon, Cheol; Yockman, James; Park, Yoon Jeong; Gitlin, Scott D; David, Allan E; Yang, Victor C

2009-11-03

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

The Multilevel Mixed Intact Group Analysis: A Mixed Method to Seek, Detect, Describe, and Explain Differences Among Intact Groups

ERIC Educational Resources Information Center

Schoonenboom, Judith

2016-01-01

Educational innovations often involve intact subgroups, such as school classes or university departments. In small-scale educational evaluation research, typically involving 1 to 20 subgroups, differences among these subgroups are often neglected. This article presents a mixed method from a qualitative perspective, in which differences among…

Structural determination of intact proteins using mass spectrometry

DOEpatents

Kruppa, Gary [San Francisco, CA; Schoeniger, Joseph S [Oakland, CA; Young, Malin M [Livermore, CA

2008-05-06

The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

Pseudomonas aeruginosa infections of intact skin.

PubMed

Agger, W A; Mardan, A

1995-02-01

Pseudomonas aeruginosa infections of healthy skin are uncommon. We report four cases of P. aeruginosa infections of intact skin. These cases illustrate the clinical spectrum of these cutaneous infections: localized, mild epidermal infections (the green nail syndrome and webbed space infections), moderately serious infections (cutaneous folliculitis and otitis externa), and, in immunocompromised patients, extremely serious infections (malignant otitis externa, perirectal infection, and ecthyma gangrenosum).

An Anatomically Resolved Mouse Brain Proteome Reveals Parkinson Disease-relevant Pathways *

PubMed Central

Choi, Jong Min; Rousseaux, Maxime W. C.; Malovannaya, Anna; Kim, Jean J.; Kutzera, Joachim; Wang, Yi; Huang, Yin; Zhu, Weimin; Maity, Suman; Zoghbi, Huda Yahya; Qin, Jun

2017-01-01

Here, we present a mouse brain protein atlas that covers 17 surgically distinct neuroanatomical regions of the adult mouse brain, each less than 1 mm3 in size. The protein expression levels are determined for 6,500 to 7,500 gene protein products from each region and over 12,000 gene protein products for the entire brain, documenting the physiological repertoire of mouse brain proteins in an anatomically resolved and comprehensive manner. We explored the utility of our spatially defined protein profiling methods in a mouse model of Parkinson's disease. We compared the proteome from a vulnerable region (substantia nigra pars compacta) of wild type and parkinsonian mice with that of an adjacent, less vulnerable, region (ventral tegmental area) and identified several proteins that exhibited both spatiotemporal- and genotype-restricted changes. We validated the most robustly altered proteins using an alternative profiling method and found that these modifications may highlight potential new pathways for future studies. This proteomic atlas is a valuable resource that offers a practical framework for investigating the molecular intricacies of normal brain function as well as regional vulnerability in neurological diseases. All of the mouse regional proteome profiling data are published on line at http://mbpa.bprc.ac.cn/. PMID:28153913

How can we conserve intact tropical peatlands?

NASA Astrophysics Data System (ADS)

Lawson, Ian; Roucoux, Katherine

2017-04-01

The scientific community has, for more than three decades, been expressing increasing alarm about the fate of peatlands in parts of Indonesia and Malaysia, where extensive land-use conversion and drainage for rice and oil palm have greatly compromised peatland hydrology, ecology, biological richness, and carbon storage. The discourse in the literature on these peatlands is now moving on from attempts to preserve the last remaining fragments of peat-swamp forest, towards discussion of how best to restore damaged ecosystems, and whether it is possible to manage plantations more 'sustainably'. It is becoming increasingly clear, however, that peatlands occur quite widely in other parts of the lowland tropics, including parts of Amazonia and the Congo Basin, and many of these peatlands can reasonably be described as 'intact': although few if any parts of the tropics are totally unaffected by human actions, the hydrology and functional ecology of these systems appear to be close to a 'natural' state. The question then arises as to what should be done with the knowledge of their existence. Here we analyse the arguments in favour of protecting intact peatlands, and the potential conflicts with other priorities such as economic development and social justice. We evaluate alternative mechanisms for protecting intact peatlands, focusing on the particular issues raised by peatlands as opposed to other kinds of tropical ecosystem. We identify ways in which natural science agendas can help to inform these arguments, using our own contributions in palaeoecology and carbon mapping as examples. Finally, we argue for a radical reconsideration of research agendas in tropical peatlands, highlighting the potential contribution of methodologies borrowed from the social sciences and humanities.

Calcium Signaling in Intact Dorsal Root Ganglia

PubMed Central

Gemes, Geza; Rigaud, Marcel; Koopmeiners, Andrew S.; Poroli, Mark J.; Zoga, Vasiliki; Hogan, Quinn H.

2013-01-01

Background Ca2+ is the dominant second messenger in primary sensory neurons. In addition, disrupted Ca2+ signaling is a prominent feature in pain models involving peripheral nerve injury. Standard cytoplasmic Ca2+ recording techniques use high K+ or field stimulation and dissociated neurons. To compare findings in intact dorsal root ganglia, we used a method of simultaneous electrophysiologic and microfluorimetric recording. Methods Dissociated neurons were loaded by bath-applied Fura-2-AM and subjected to field stimulation. Alternatively, we adapted a technique in which neuronal somata of intact ganglia were loaded with Fura-2 through an intracellular microelectrode that provided simultaneous membrane potential recording during activation by action potentials (APs) conducted from attached dorsal roots. Results Field stimulation at levels necessary to activate neurons generated bath pH changes through electrolysis and failed to predictably drive neurons with AP trains. In the intact ganglion technique, single APs produced measurable Ca2+ transients that were fourfold larger in presumed nociceptive C-type neurons than in nonnociceptive Aβ-type neurons. Unitary Ca2+ transients summated during AP trains, forming transients with amplitudes that were highly dependent on stimulation frequency. Each neuron was tuned to a preferred frequency at which transient amplitude was maximal. Transients predominantly exhibited monoexponential recovery and had sustained plateaus during recovery only with trains of more than 100 APs. Nerve injury decreased Ca2+ transients in C-type neurons, but increased transients in Aβ-type neurons. Conclusions Refined observation of Ca2+ signaling is possible through natural activation by conducted APs in undissociated sensory neurons and reveals features distinct to neuronal types and injury state. PMID:20526180

HENMT1 and piRNA Stability Are Required for Adult Male Germ Cell Transposon Repression and to Define the Spermatogenic Program in the Mouse

PubMed Central

Lim, Shu Ly; Geoghegan, Joel; Hempfling, Anna-Lena; Bergmann, Martin; Goodnow, Christopher C.; Ormandy, Christopher J.; Wong, Lee; Mann, Jeff; Scott, Hamish S.; Jamsai, Duangporn; Adelson, David L.

2015-01-01

piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2’ O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program. PMID:26496356

Losing a jewel-Rapid declines in Myanmar's intact forests from 2002-2014.

PubMed

Bhagwat, Tejas; Hess, Andrea; Horning, Ned; Khaing, Thiri; Thein, Zaw Min; Aung, Kyaw Moe; Aung, Kyaw Htet; Phyo, Paing; Tun, Ye Lin; Oo, Aung Htat; Neil, Anthony; Thu, Win Myo; Songer, Melissa; LaJeunesse Connette, Katherine; Bernd, Asja; Huang, Qiongyu; Connette, Grant; Leimgruber, Peter

2017-01-01

New and rapid political and economic changes in Myanmar are increasing the pressures on the country's forests. Yet, little is known about the past and current condition of these forests and how fast they are declining. We mapped forest cover in Myanmar through a consortium of international organizations and environmental non-governmental groups, using freely-available public domain data and open source software tools. We used Landsat satellite imagery to assess the condition and spatial distribution of Myanmar's intact and degraded forests with special focus on changes in intact forest between 2002 and 2014. We found that forests cover 42,365,729 ha or 63% of Myanmar, making it one of the most forested countries in the region. However, severe logging, expanding plantations, and degradation pose increasing threats. Only 38% of the country's forests can be considered intact with canopy cover >80%. Between 2002 and 2014, intact forests declined at a rate of 0.94% annually, totaling more than 2 million ha forest loss. Losses can be extremely high locally and we identified 9 townships as forest conversion hotspots. We also delineated 13 large (>100,000 ha) and contiguous intact forest landscapes, which are dispersed across Myanmar. The Northern Forest Complex supports four of these landscapes, totaling over 6.1 million ha of intact forest, followed by the Southern Forest Complex with three landscapes, comprising 1.5 million ha. These remaining contiguous forest landscape should have high priority for protection. Our project demonstrates how open source data and software can be used to develop and share critical information on forests when such data are not readily available elsewhere. We provide all data, code, and outputs freely via the internet at (for scripts: https://bitbucket.org/rsbiodiv/; for the data: http://geonode.themimu.info/layers/geonode%3Amyan_lvl2_smoothed_dec2015_resamp).

Effect of cyanotoxins on the hypothalamic-pituitary-gonadal axis in male adult mouse.

PubMed

Xiong, Xiaolu; Zhong, Anyuan; Xu, Huajun

2014-01-01

Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic-Pituitary-Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis. Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice. MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.

Development and function of human innate immune cells in a humanized mouse model.

PubMed

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

2014-04-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

Development and function of human innate immune cells in a humanized mouse model

PubMed Central

Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V.; Teichmann, Lino L.; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A. Karolina; Manz, Markus G.; Flavell, Richard A.

2014-01-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology. PMID:24633240

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

PubMed

Khan, Kainat; Singh, Akanksha; Mittal, Monika; Sharan, Kunal; Singh, Nidhi; Dixit, Preety; Sanyal, Sabyasachi; Maurya, Rakesh; Chattopadhyay, Naibedya

2012-12-01

[6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated. The effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol. Daily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

PubMed Central

Ufimtseva, Elena

2016-01-01

The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells. PMID:27066505

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro.

PubMed

Jung, Song-yi; Willard, Scott T

2014-01-30

The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

PubMed Central

2014-01-01

Background The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. Methods We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. Results The level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin. Conclusions The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a

The development of inter-strain variation in cortical and trabecular traits during growth of the mouse lumbar vertebral body.

PubMed

Ramcharan, M A; Faillace, M E; Guengerich, Z; Williams, V A; Jepsen, K J

2017-03-01

How cortical and trabecular bone co-develop to establish a mechanically functional structure is not well understood. Comparing early postnatal differences in morphology of lumbar vertebral bodies for three inbred mouse strains identified coordinated changes within and between cortical and trabecular traits. These early coordinate changes defined the phenotypic differences among the inbred mouse strains. Age-related changes in cortical and trabecular traits have been well studied; however, very little is known about how these bone tissues co-develop from day 1 of postnatal growth to establish functional structures by adulthood. In this study, we aimed to establish how cortical and trabecular tissues within the lumbar vertebral body change during growth for three inbred mouse strains that express wide variation in adult bone structure and function. Bone traits were quantified for lumbar vertebral bodies of female A/J, C57BL/6J (B6), and C3H/HeJ (C3H) inbred mouse strains from 1 to 105 days of age (n = 6-10 mice/age/strain). Inter-strain differences in external bone size were observed as early as 1 day of age. Reciprocal and rapid changes in the trabecular bone volume fraction and alignment in the direction of axial compression were observed by 7 days of age. Importantly, the inter-strain difference in adult trabecular bone volume fraction was established by 7 days of age. Early variation in external bone size and trabecular architecture was followed by progressive increases in cortical area between 28 and 105 days of age, with the greatest increases in cortical area seen in the mouse strain with the lowest trabecular mass. Establishing the temporal changes in bone morphology for three inbred mouse strains revealed that genetic variation in adult trabecular traits were established early in postnatal development. Early variation in trabecular architecture preceded strain-specific increases in cortical area and changes in cortical thickness. This study

Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

NASA Astrophysics Data System (ADS)

Candeo, Alessia; Sana, Ilenia; Ferrari, Eleonora; Maiuri, Luigi; D'Andrea, Cosimo; Valentini, Gianluca; Bassi, Andrea

2016-05-01

Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.

Dissipation of the Proton Electrochemical Potential in Intact and Lysed Chloroplasts 1

PubMed Central

Nishio, John N.; Whitmarsh, John

1991-01-01

Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts. PMID:16668015

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

PubMed Central

Yu, Zhi-Bin; Wei, Hongguang

2012-01-01

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213–C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97–H105, 2010). Here we characterized Ca2+-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca2+ was unchanged, but the peak Ca2+ transient was lowered and the durations of Ca2+ rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca2+ transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca2+, resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences. PMID:22538236

Childrearing Fathers in Intact Families With Preschoolers.

ERIC Educational Resources Information Center

Radin, Norma

Few empirical studies have specifically examined antecedents and consequences of reversing childrearing roles. A sample of 59 white, intact, middle-class families with a preschool-aged child, 32 with boys and 27 with girls, was studied. Also explored were the father's sex-role orientation and selected paternal behaviors and attitudes. It was found…

Multi-Axis Prosthetic Knee Resembles Alpine Skiing Movements of an Intact Leg

PubMed Central

Demšar, Ivan; Duhovnik, Jože; Lešnik, Blaž; Supej, Matej

2015-01-01

The purpose of the study was to analyse the flexion angles of the ski boot, ankle and knee joints of an above-knee prosthesis and to compare them with an intact leg and a control group of skiers. One subject with an above-knee amputation of the right leg and eight healthy subjects simulated the movement of a skiing turn by performing two-leg squats in laboratory conditions. By adding additional loads in proportion to body weight (BW; +1/3 BW, +2/3 BW, +3/3 BW), various skiing regimes were simulated. Change of Flexion Angle (CoFA) and Range of Motion (RoM) in the ski boot, ankle and knee joints were calculated and compared. An average RoM in the skiing boot on the side of prosthesis (4.4 ± 1.1°) was significantly lower compared to an intact leg (5.9 ± 1.8°) and the control group (6.5 ± 2.3°). In the ankle joint, the average RoM was determined to be 13.2±2.9° in the prosthesis, 12.7 ± 2.8° in an intact leg and 14.8±3.6 in the control group. However, the RoM of the knee joint in the prosthesis (42.2 ± 4.2°) was significantly larger than that of the intact leg (34.7 ± 4.4°). The average RoM of the knee joint in the control group was 47.8 ± 5.4°. The influences of additional loads on the kinematics of the lower extremities were different on the side of the prosthesis and on the intact leg. In contrast, additional loads did not produce any significant differences in the control group. Although different CoFAs in the ski boot, ankle and knee joints were used, an above-knee prosthesis with a built-in multi-axis prosthetic knee enables comparable leg kinematics in simulated alpine skiing. Key points The RoM in the ski boot on the side of the prosthetic leg was smaller than the RoM of the intact leg and the control group of healthy subjects. The RoM in the ankle joint of prosthetic leg was comparable to that of the intact leg and the control group of healthy subjects. The RoM in the prosthetic knee joint was greater than the RoM in the knee joint of the

Vehicle influence on permeation through intact and compromised skin.

PubMed

Gujjar, Meera; Banga, Ajay K

2014-09-10

The purpose of this study was to compare the transdermal permeation of a model compound, diclofenac diethylamine, from a hydrophilic and lipophilic vehicle across in vitro models simulating compromised skin. Mineral oil served as a lipophilic vehicle while 10mM phosphate buffered saline served as a hydrophilic vehicle. Compromised skin was simulated by tape stripping, delipidization, or microneedle application and compared with intact skin as a control. Transepidermal water loss was measured to assess barrier function. Skin compromised with tape stripping and delipidization significantly (p<0.05) increased permeation of diclofenac diethylamine compared to intact and microneedle treated skin with phosphate buffered saline vehicle. A similar trend in permeation was observed with mineral oil as the vehicle. For both vehicles, permeation across skin increased in the same order and correlated with degree of barrier impairment as indicated by transepidermal water loss values: intact

Antimicrobial activity of a new intact skin antisepsis formulation.

PubMed

Russo, Antonello; Viotti, Pier Luigi; Vitali, Matteo; Clementi, Massimo

2003-04-01

Different antiseptic formulations have shown limitations when applied to disinfecting intact skin, notably short-term tolerability and/or efficacy. The purpose of this study was optimizing a new antiseptic formulation specifically targeted at intact skin disinfection and evaluating its in vitro microbicidal activity and in vivo efficacy. The biocidal properties of the antiseptic solution containing 0.5% chloramine-T diluted in 50% isopropyl alcohol (Cloral; Eurospital SpA Trieste, Italy) were measured in vitro versus gram-positive-, gram-negative-, and acid-alcohol-resistant germs and fungi with standard suspension tests in the presence of fetal bovine serum. Virus-inhibiting activity was evaluated in vitro against human cytomegalovirus, herpes simplex virus, poliovirus, hepatitis B virus, and hepatitis C virus. Tests used different methods for the different biologic and in vitro replication capacity of these human viruses. Lastly, Cloral tolerability and skin colonization retardation efficacy after disinfection were studied in vivo. The antiseptic under review showed fast and sustained antimicrobial activity. The efficacy of Cloral against clinically important bacterial and viral pathogens and fungi was highlighted under the experimental conditions described in this article. Finally, microbial regrowth lag and no side effects were documented in vivo after disinfection of 11 volunteers. A stable chloramine-T solution in isopropyl alcohol may be suggested for intact skin antisepsis.

Patterning by heritage in mouse molar row development.

PubMed

Prochazka, Jan; Pantalacci, Sophie; Churava, Svatava; Rothova, Michaela; Lambert, Anne; Lesot, Hervé; Klein, Ophir; Peterka, Miroslav; Laudet, Vincent; Peterkova, Renata

2010-08-31

It is known from paleontology studies that two premolars have been lost during mouse evolution. During mouse mandible development, two bud-like structures transiently form that may represent rudimentary precursors of the lost premolars. However, the interpretation of these structures and their significance for mouse molar development are highly controversial because of a lack of molecular data. Here, we searched for typical tooth signaling centers in these two bud-like structures, and followed their fate using molecular markers, 3D reconstructions, and lineage tracing in vitro. Transient signaling centers were indeed found to be located at the tips of both the anterior and posterior rudimentary buds. These centers expressed a similar set of molecular markers as the "primary enamel knot" (pEK), the signaling center of the first molar (M1). These two transient signaling centers were sequentially patterned before and anterior to the M1 pEK. We also determined the dynamics of the M1 pEK, which, slightly later during development, spread up to the field formerly occupied by the posterior transient signaling center. It can be concluded that two rudimentary tooth buds initiate the sequential development of the mouse molars and these have previously been mistaken for early stages of M1 development. Although neither rudiment progresses to form an adult tooth, the posterior one merges with the adjacent M1, which may explain the anterior enlargement of the M1 during mouse family evolution. This study highlights how rudiments of lost structures can stay integrated and participate in morphogenesis of functional organs and help in understanding their evolution, as Darwin suspected long ago.

GESTATIONAL EXPOSURE TO ETHANE DIMETHANESULFONATE PERMANENTLY ALTERS REPRODUCTIVE COMPETENCE IN THE CD-1 MOUSE

EPA Science Inventory

While the adult mouse Leydig cell (LC) has been considered refractory to cytotoxic destruction by ethane dimethanesulfonate (EDS), the potential consequences of exposure during reproductive development in this species are unknown. Herein pregnant CD-1 mice were treated with 160 m...

Hericium erinaceus Extract Reduces Anxiety and Depressive Behaviors by Promoting Hippocampal Neurogenesis in the Adult Mouse Brain.

PubMed

Ryu, Sun; Kim, Hyoun Geun; Kim, Joo Youn; Kim, Seong Yun; Cho, Kyung-Ok

2018-02-01

Versatile biological activities of Hericium erinaceus (HE) have been reported in many brain diseases. However, roles of HE in major psychiatric disorders such as depression and anxiety remain to be investigated. Therefore, we evaluated whether HE could reduce anxiety and depressive behaviors in the adult mouse and its underlying mechanisms. Male C57BL/6 mice were administered HE (20 or 60 mg/kg, p.o.) or saline once a day for 4 weeks. Open field and tail suspension tests were performed 30 min after the last administration of HE, followed by forced swim test 2 days later. We found that chronic administration of HE showed anxiolytic and antidepressant-like effects. To elucidate possible mechanisms, proliferative activity of the hippocampal progenitor cells was assessed by immunohistochemistry of proliferating cell nuclear antigen (PCNA) and Ki67. Moreover, to evaluate neuronal survival in the dentate gyrus, 5-bromo-2'-deoxyuridine (BrdU) (120 mg/kg, i.p.) was given at the first day of HE administration, followed by isolation of the brains 4 weeks later. HE (60 mg/kg) increased the number of PCNA- and Ki67-positive cells in the subgranular zone of the hippocampus, indicating increased proliferation of hippocampal progenitors. In addition, BrdU- and BrdU/NeuN-positive cells in the dentate gyrus were significantly increased when treated with HE (60 mg/kg) compared with the saline-treated group, demonstrating enhanced neurogenesis by HE treatment. Taken together, the results indicate that chronic HE administration can exert anxiolytic and antidepressant-like effects, possibly by enhancing adult hippocampal neurogenesis.

Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

PubMed Central

Clerc, Pascaline; Polster, Brian M.

2012-01-01

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria. PMID:22496810

The scarless heart and the MRL mouse.

PubMed

Heber-Katz, Ellen; Leferovich, John; Bedelbaeva, Khamilia; Gourevitch, Dmitri; Clark, Lise

2004-05-29

The ability to regenerate tissues and limbs in its most robust form is seen in many non-mammalian species. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivalling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. The adult MRL mouse regrows cartilage, skin, hair follicles and myocardium with near perfect fidelity and without scarring. This is seen in the ability to close through-and-through ear holes, which are generally used for lifelong identification of mice, and the anatomic and functional recovery of myocardium after a severe cryo-injury. We present histological, biochemical and genetic data indicating that the enhanced breakdown of scar-like tissue may be an underlying factor in the MRL regenerative response. Studies as to the source of the cells in the regenerating MRL tissue are discussed. Such studies appear to support multiple mechanisms for cell replacement.

Age-Dependent Long-Term Potentiation Deficits in the Prefrontal Cortex of the Fmr1 Knockout Mouse Model of Fragile X Syndrome.

PubMed

Martin, Henry G S; Lassalle, Olivier; Brown, Jonathan T; Manzoni, Olivier J

2016-05-01

The most common inherited monogenetic cause of intellectual disability is Fragile X syndrome (FXS). The clinical symptoms of FXS evolve with age during adulthood; however, neurophysiological data exploring this phenomenon are limited. The Fmr1 knockout (Fmr1KO) mouse models FXS, but studies in these mice of prefrontal cortex (PFC) function are underrepresented, and aging linked data are absent. We studied synaptic physiology and activity-dependent synaptic plasticity in the medial PFC of Fmr1KO mice from 2 to 12 months. In young adult Fmr1KO mice, NMDA receptor (NMDAR)-mediated long-term potentiation (LTP) is intact; however, in 12-month-old mice this LTP is impaired. In parallel, there was an increase in the AMPAR/NMDAR ratio and a concomitant decrease of synaptic NMDAR currents in 12-month-old Fmr1KO mice. We found that acute pharmacological blockade of mGlu5 receptor in 12-month-old Fmr1KO mice restored a normal AMPAR/NMDAR ratio and LTP. Taken together, the data reveal an age-dependent deficit in LTP in Fmr1KO mice, which may correlate to some of the complex age-related deficits in FXS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetic Rescue of Glycosylation-deficient Fgf23 in the Galnt3 Knockout Mouse

PubMed Central

Gray, Amie K.; Padgett, Leah R.; Allen, Matthew R.; Clinkenbeard, Erica L.; Sarpa, Nicole M.; White, Kenneth E.; Econs, Michael J.

2014-01-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence (176RHTR179↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus in

Genetic rescue of glycosylation-deficient Fgf23 in the Galnt3 knockout mouse.

PubMed

Ichikawa, Shoji; Gray, Amie K; Padgett, Leah R; Allen, Matthew R; Clinkenbeard, Erica L; Sarpa, Nicole M; White, Kenneth E; Econs, Michael J

2014-10-01

Fibroblast growth factor 23 (FGF23) is a hormone that inhibits renal phosphate reabsorption and 1,25-dihydroxyvitamin D biosynthesis. The FGF23 subtilisin-like proprotein convertase recognition sequence ((176)RHTR(179)↓) is protected by O-glycosylation through ppGalNAc-T3 (GALNT3) activity. Thus, inactivating GALNT3 mutations render FGF23 susceptible to proteolysis, thereby reducing circulating intact hormone levels and leading to hyperphosphatemic familial tumoral calcinosis. To further delineate the role of glycosylation in the Fgf23 function, we generated an inducible FGF23 transgenic mouse expressing human mutant FGF23 (R176Q and R179Q) found in patients with autosomal dominant hypophosphatemic rickets (ADHR) and bred this animal to Galnt3 knockout mice, a model of familial tumoral calcinosis. Due to the low intact Fgf23 level, Galnt3 knockout mice with wild-type Fgf23 alleles were hyperphosphatemic. In contrast, carriers of the mutant FGF23 transgene, regardless of Galnt3 mutation status, had significantly higher serum intact FGF23, resulting in severe hypophosphatemia. Importantly, serum phosphorus and FGF23 were comparable between transgenic mice with or without normal Galnt3 alleles. To determine whether the presence of the ADHR mutation could improve biochemical and skeletal abnormalities in Galnt3-null mice, these mice were also mated to Fgf23 knock-in mice, carrying heterozygous or homozygous R176Q ADHR Fgf23 mutations. The knock-in mice with functional Galnt3 had normal Fgf23 but were slightly hypophosphatemic. The stabilized Fgf23 ADHR allele reversed the Galnt3-null phenotype and normalized total Fgf23, serum phosphorus, and bone Fgf23 mRNA. However, the skeletal phenotype was unaffected. In summary, these data demonstrate that O-glycosylation by ppGaINAc-T3 is only necessary for proper secretion of intact Fgf23 and, once secreted, does not affect Fgf23 function. Furthermore, the more stable Fgf23 ADHR mutant protein could normalize serum phosphorus

Centralized mouse repositories.

PubMed

Donahue, Leah Rae; Hrabe de Angelis, Martin; Hagn, Michael; Franklin, Craig; Lloyd, K C Kent; Magnuson, Terry; McKerlie, Colin; Nakagata, Naomi; Obata, Yuichi; Read, Stuart; Wurst, Wolfgang; Hörlein, Andreas; Davisson, Muriel T

2012-10-01

Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

PubMed

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

Comparison of Ripening Processes in Intact Tomato Fruit and Excised Pericarp Discs 1

PubMed Central

Campbell, Alan D.; Huysamer, Marius; Stotz, Henrik U.; Greve, L. Carl; Labavitch, John M.

1990-01-01

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L*a*b* color space, changing from green (a* < −5) to red (a* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO2 production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a* = −5), appeared as discs turned yellow-red (a* = 5), and increased another eight-fold as discs became full red (a* value +20). Carboxymethyl-cellulase activity, low in extracts from

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis

NASA Astrophysics Data System (ADS)

Hellebust, Anne; Rosbach, Kelsey; Wu, Jessica Keren; Nguyen, Jennifer; Gillenwater, Ann; Vigneswaran, Nadarajah; Richards-Kortum, Rebecca

2013-12-01

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

Can assisted reproductive technologies cause adult-onset disease? Evidence from human and mouse

PubMed Central

Vrooman, Lisa A.; Bartolomei, Marisa S.

2016-01-01

Millions of children have been born worldwide though assisted reproductive technologies (ART). Consistent with the Developmental Origins of Health and Disease hypothesis, there is concern that ART can induce adverse effects, especially because procedures coincide with epigenetic reprogramming events. Although the majority of studies investigating the effects of ART have focused on perinatal outcomes, more recent studies demonstrate that ART-conceived children may be at increased risk for postnatal effects. Here, we present the current epidemiological evidence that ART-conceived children have detectable differences in blood pressure, body composition, and glucose homeostasis. Similar effects are observed in the ART mouse model, which have no underlying infertility, suggesting that cardiometabolic effects are likely caused by ART procedures and not due to reasons related to infertility. We propose that the mouse system can, consequently, be used to adequately study, modify, and improve outcomes for ART children. PMID:27474254

Mouse embryonic head as a site for hematopoietic stem cell development.

PubMed

Li, Zhuan; Lan, Yu; He, Wenyan; Chen, Dongbo; Wang, Jun; Zhou, Fan; Wang, Yu; Sun, Huayan; Chen, Xianda; Xu, Chunhong; Li, Sha; Pang, Yakun; Zhang, Guangzhou; Yang, Liping; Zhu, Lingling; Fan, Ming; Shang, Aijia; Ju, Zhenyu; Luo, Lingfei; Ding, Yuqiang; Guo, Wei; Yuan, Weiping; Yang, Xiao; Liu, Bing

2012-11-02

In the mouse embryo, the aorta-gonad-mesonephros (AGM) region is considered to be the sole location for intraembryonic emergence of hematopoietic stem cells (HSCs). Here we report that, in parallel to the AGM region, the E10.5-E11.5 mouse head harbors bona fide HSCs, as defined by long-term, high-level, multilineage reconstitution and self-renewal capacity in adult recipients, before HSCs enter the circulation. The presence of hemogenesis in the midgestation head is indicated by the appearance of intravascular cluster cells and the blood-forming capacity of a sorted endothelial cell population. In addition, lineage tracing via an inducible VE-cadherin-Cre transgene demonstrates the hemogenic capacity of head endothelium. Most importantly, a spatially restricted lineage labeling system reveals the physiological contribution of cerebrovascular endothelium to postnatal HSCs and multilineage hematopoiesis. We conclude that the mouse embryonic head is a previously unappreciated site for HSC emergence within the developing embryo. Copyright © 2012 Elsevier Inc. All rights reserved.

Successful mouse cloning of an outbred strain by trichostatin A treatment after somatic nuclear transfer.

PubMed

Kishigami, Satoshi; Bui, Hong-Thuy; Wakayama, Sayaka; Tokunaga, Kenzo; Van Thuan, Nguyen; Hikichi, Takafusa; Mizutani, Eiji; Ohta, Hiroshi; Suetsugu, Rinako; Sata, Tetsutaro; Wakayama, Teruhiko

2007-02-01

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, extremely low success rates have plagued this technique for a decade. Further in mice, the "clonable" strains have been limited to mainly hybrid F1 strains such as B6D2F1. Recently, we established a new efficient cloning technique using trichostatin A (TSA) which leads to a 2-5 fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. To further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4-5% success rates, which is comparable to 5-7% of B6D2F1. Thus, the TSA treatment is the first cloning technique to allow us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from hybrid strains, but also enables mouse cloning from normally "unclonable" strains.

Centralized Mouse Repositories

PubMed Central

Donahue, Leah Rae; de Angelis, Martin Hrabe; Hagn, Michael; Franklin, Craig; Lloyd, K. C. Kent; Magnuson, Terry; McKerlie, Colin; Nakagata, Naomi; Obata, Yuichi; Read, Stuart; Wurst, Wolfgang; Hörlein, Andreas; Davisson, Muriel T.

2013-01-01

Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world. PMID:22945696

Dehydration and clearing of adult Drosophila for ultramicroscopy.

PubMed

Becker, Klaus; Jährling, Nina; Saghafi, Saiedeh; Dodt, Hans-Ulrich

2013-07-01

This protocol describes the preparation of adult flies for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. Thus, specimens for UM need to be sufficiently transparent, which requires chemical clearing in most cases. In this protocol, Drosophila melanogaster adults are fixed, dehydrated in ethanol, and then cleared in a solution of benzyl alcohol and benzyl benzoate.

Adult Children of Divorce: Curative Factors of Support Group Therapy.

ERIC Educational Resources Information Center

McGuire, Kent Allen

There is substantial evidence that for some children whose parents divorce, long-term effects of divorce may be with them into adulthood. Compared to individuals from intact families, adult children of divorce tend to experience an increased level of depression, anger, hostility, sadness, sorrow, and anxiety; an increased vulnerability to stress;…

Consequences of Parental Divorce and Marital Unhappiness for Adult Well-Being.

ERIC Educational Resources Information Center

Amato, Paul R.; Booth, Alan

1991-01-01

Among 1,243 adults nationwide, individuals who experienced parental divorce as children scored lower than those from happily intact families of origin on measures of psychological, social, and marital well-being. Multiple parental divorces and divorces involving deterioration of parent-child relations appeared particularly problematic. Contains 39…

(Photosynthesis in intact plants)

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

Not Available

Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allowmore » us to explore new options in the attempt to understand function at the level of molecular structure.« less

Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition.

PubMed

Goldfarb, Ilona Telefus; Adeli, Sharareh; Berk, Tucker; Phillippe, Mark

2018-05-01

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells. Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein). Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells. DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina.

PubMed

Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B

2014-04-30

A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors.

Gender markedly modulates behavioral thermoregulation in a non-human primate species, the mouse lemur (Microcebus murinus).

PubMed

Terrien, J; Perret, M; Aujard, F

2010-11-02

Age and gender are known to significantly modulate thermoregulatory capacities in mammals, suggesting strong impacts on behavioral adjustments, which are used to minimize the energy costs of thermoregulation. We tested the effects of sex and age on spontaneous choice of ambient temperature (Ta) in a non-human primate species, the mouse lemur (Microcebus murinus). The animals acclimated to both winter and summer photoperiods, two seasons significantly modifying thermoregulation function, were experimented in a thermal gradient device. During winter, adult males did not show preference for warm Tas whereas old males did. In contrast, female mouse lemurs of both age categories exhibited great preferences for warm Tas. Acclimation to summer revealed that males selected colder Ta for the day than during the night. Such behavior did not exist in females. Old females explored and selected warmer nests than adult ones. This study raised novel issues on the effect of gender on thermoregulatory capacities in the mouse lemur. Females probably use behavioral adjustments to limit energy expenditure and might prefer to preserve energy for maternal investment by anticipation of and during the breeding season. Further experiments focusing on female thermoregulatory capacities are needed to better understand the energy challenge that may occur during winter and summer in female mouse lemurs, and whether this trade-off changes during aging. Copyright © 2010 Elsevier Inc. All rights reserved.

Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets.

PubMed Central

Kawaguchi, S

1989-01-01

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies. PMID:2467876

Functional abilities in older adults with mild cognitive impairment.

PubMed

Burton, Catherine L; Strauss, Esther; Bunce, David; Hunter, Michael A; Hultsch, David F

2009-01-01

A classification scheme and general set of criteria for diagnosing mild cognitive impairment (MCI) were recently proposed by a multidisciplinary group of experts who met at an international symposium on MCI. One of the proposed criteria included preserved basic activities of daily living and minimal impairment in complex instrumental activities of daily living (IADLs). To investigate whether older adults with MCI classified according to the subtypes identified by the Working Group (i.e. amnestic, single non-memory domain, and multiple domain with or without a memory component) differed from cognitively intact older adults on a variety of measures indexing IADLs and to examine how well measures of IADL predict concurrent MCI status. Two hundred and fifty community-dwelling older adults, ranging in age from 66 to 92, completed self-report measures of IADLs (Lawton and Brody IADL Scale, Scales of Independent Behaviour-Revised--SIB-R) and a measure of everyday problem solving indexing IADLs (Everyday Problems Test--EPT). Ratings of participants' IADL functioning were also obtained from informants (e.g. spouse, adult child and friend). Older adults with multiple-domain MCI demonstrated poorer IADL functioning than older adults with no cognitive impairment on the EPT and the SIB-R (both self- and informant-report versions). The multiple-domain MCI participants also demonstrated poorer IADLs than MCI participants with impairments in a single cognitive domain on the self-reported SIB-R and EPT. The single-domain MCI groups demonstrated poorer IADLs than older adults without cognitive impairment on the informant-reported SIB-R and EPT. No significant group differences were found on the Lawton and Brody IADL Scale. Using the EPT and SIB-R as predictors in a multinomial regression analysis, MCI group status was reliably predicted, but the classification rate was poor. Individuals with MCI demonstrated poorer IADL functioning compared to cognitively intact older adults

Identification of a set of genes showing regionally enriched expression in the mouse brain

PubMed Central

D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM

2008-01-01

Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression. PMID:18625066

Identification of a set of genes showing regionally enriched expression in the mouse brain.

PubMed

D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa L C; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven J M

2008-07-14

The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.

The Behavior and Acrosomal Status of Mouse Spermatozoa In Vitro, and Within the Oviduct During Fertilization after Natural Mating.

PubMed

Hino, Toshiaki; Muro, Yuko; Tamura-Nakano, Miwa; Okabe, Masaru; Tateno, Hiroyuki; Yanagimachi, Ryuzo

2016-09-01

Although 90%-100% of mouse oocytes can be fertilized in vitro with capacitated spermatozoa within 1 h after insemination, oocytes within the oviduct are fertilized one by one over a period of several hours. In vitro experiments showed that both acrosome-intact and acrosome-reacted spermatozoa entered the cumulus oophorus, but that acrosome-reacted spermatozoa reached the surface of oocytes more readily than acrosome-intact spermatozoa. During the period of fertilization within the oviduct, acrosome-reacted spermatozoa were seen throughout the isthmus, but with higher incidence in the upper than in the mid- and lower segments of the isthmus. Very few spermatozoa were present in the ampulla, and almost all were acrosome reacted. Although the cumulus oophorus and zona pellucida are known to be able to induce or facilitate the acrosome reaction of spermatozoa, this picture makes it likely that almost all fertilizing mouse spermatozoa within the oviduct begin to react before ascending from the isthmus to the ampulla. We witnessed a reacted spermatozoon that stayed on the zona pellucida of a fertilized oocyte for a while; it then moved out of the cumulus before reaching the zona pellucida of the nearby unfertilized oocyte. We noted that only a few spermatozoa migrate from the isthmus to the ampulla during the progression of fertilization, and this must be one of the reasons why we do not see many spermatozoa swarming around a single oocyte during in vivo fertilization. © 2016 by the Society for the Study of Reproduction, Inc.

Killing and caching of an adult White-tailed deer, Odocoileus virginianus, by a single Gray Wolf, Canis lupus

USGS Publications Warehouse

Nelson, Michael E.

2011-01-01

A single Gray Wolf (Canis lupus) killed an adult male White-tailed Deer (Odocoileus virginianus) and cached the intact carcass in 76 cm of snow. The carcass was revisited and entirely consumed between four and seven days later. This is the first recorded observation of a Gray Wolf caching an entire adult deer.

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Isolation Efficiency of Mouse Pancreatic Stem Cells Is Age Dependent.

PubMed

Kuise, Takashi; Noguchi, Hirofumi; Saitoh, Issei; Kataoka, Hitomi Usui; Watanabe, Masami; Noguchi, Yasufumi; Fujiwara, Toshiyoshi

2013-11-10

Mouse pancreatic stem cells have been isolated from mouse pancreata. This study evaluated the efficacy of isolating mouse pancreatic stem cells using mice of different ages. The pancreata of newborn mice, 8-week-old mice, and 24-week-old mice were harvested and digested by using collagenase. The "duct-like" cells in the digested pancreatic tissue were then inoculated into 96-well plates, cloned by limiting dilution, and cultured in DMEM with 20% FBS. Pancreatic stem cells were isolated from the pancreata of all newborn mice, while cells could only be isolated from 10% of the pancreata of 8-week-old mice and could not be isolated from the pancreata of any 24-week-old mice. These data suggest that young mice may have some pancreatic stem cells and that older mice may only have a few pancreatic stem cells. These data also indicate that it is extremely difficult to isolate pancreatic stem cells from older mice, suggesting that future research focus its efforts on finding methods of isolating pancreatic stem cells from adult mice.

A Cell Line Producing Recombinant Nerve Growth Factor Evokes Growth Responses in Intrinsic and Grafted Central Cholinergic Neurons

NASA Astrophysics Data System (ADS)

Ernfors, Patrik; Ebendal, Ted; Olson, Lars; Mouton, Peter; Stromberg, Ingrid; Persson, Hakan

1989-06-01

The rat β nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

Acute detachment of hexokinase II from mitochondria modestly increases oxygen consumption of the intact mouse heart.

PubMed

Nederlof, Rianne; Denis, Simone; Lauzier, Benjamin; Rosiers, Christine Des; Laakso, Markku; Hagen, Jacob; Argmann, Carmen; Wanders, Ronald; Houtkooper, Riekelt H; Hollmann, Markus W; Houten, Sander M; Zuurbier, Coert J

2017-07-01

Cardiac hexokinase II (HKII) can translocate between cytosol and mitochondria and change its cellular expression with pathologies such as ischemia-reperfusion, diabetes and heart failure. The cardiac metabolic consequences of these changes are unknown. Here we measured energy substrate utilization in cytosol and mitochondria using stabile isotopes and oxygen consumption of the intact perfused heart for 1) an acute decrease in mitochondrial HKII (mtHKII), and 2) a chronic decrease in total cellular HKII. We first examined effects of 200nM TAT (Trans-Activator of Transcription)-HKII peptide treatment, which was previously shown to acutely decrease mtHKII by ~30%. In Langendorff-perfused hearts TAT-HKII resulted in a modest, but significant, increased oxygen consumption, while cardiac performance was unchanged. At the metabolic level, there was a nonsignificant (p=0.076) ~40% decrease in glucose contribution to pyruvate and lactate formation through glycolysis and to mitochondrial citrate synthase flux (6.6±1.1 vs. 11.2±2.2%), and an 35% increase in tissue pyruvate (27±2 vs. 20±2pmol/mg; p=0.033). Secondly, we compared WT and HKII +/- hearts (50% chronic decrease in total HKII). RNA sequencing revealed no differential gene expression between WT and HKII +/- hearts indicating an absence of metabolic reprogramming at the transcriptional level. Langendorff-perfused hearts showed no significant differences in glycolysis (0.34±0.03μmol/min), glucose contribution to citrate synthase flux (35±2.3%), palmitate contribution to citrate synthase flux (20±1.1%), oxygen consumption or mechanical performance between WT and HKII +/- hearts. These results indicate that acute albeit not chronic changes in mitochondrial HKII modestly affect cardiac oxygen consumption and energy substrate metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Losing a jewel—Rapid declines in Myanmar’s intact forests from 2002-2014

PubMed Central

Horning, Ned; Khaing, Thiri; Thein, Zaw Min; Aung, Kyaw Moe; Aung, Kyaw Htet; Phyo, Paing; Tun, Ye Lin; Oo, Aung Htat; Neil, Anthony; Thu, Win Myo; Songer, Melissa; Huang, Qiongyu; Connette, Grant; Leimgruber, Peter

2017-01-01

New and rapid political and economic changes in Myanmar are increasing the pressures on the country’s forests. Yet, little is known about the past and current condition of these forests and how fast they are declining. We mapped forest cover in Myanmar through a consortium of international organizations and environmental non-governmental groups, using freely-available public domain data and open source software tools. We used Landsat satellite imagery to assess the condition and spatial distribution of Myanmar’s intact and degraded forests with special focus on changes in intact forest between 2002 and 2014. We found that forests cover 42,365,729 ha or 63% of Myanmar, making it one of the most forested countries in the region. However, severe logging, expanding plantations, and degradation pose increasing threats. Only 38% of the country’s forests can be considered intact with canopy cover >80%. Between 2002 and 2014, intact forests declined at a rate of 0.94% annually, totaling more than 2 million ha forest loss. Losses can be extremely high locally and we identified 9 townships as forest conversion hotspots. We also delineated 13 large (>100,000 ha) and contiguous intact forest landscapes, which are dispersed across Myanmar. The Northern Forest Complex supports four of these landscapes, totaling over 6.1 million ha of intact forest, followed by the Southern Forest Complex with three landscapes, comprising 1.5 million ha. These remaining contiguous forest landscape should have high priority for protection. Our project demonstrates how open source data and software can be used to develop and share critical information on forests when such data are not readily available elsewhere. We provide all data, code, and outputs freely via the internet at (for scripts: https://bitbucket.org/rsbiodiv/; for the data: http://geonode.themimu.info/layers/geonode%3Amyan_lvl2_smoothed_dec2015_resamp) PMID:28520726

Adult mouse motor units develop almost all of their force in the subprimary range: a new all-or-none strategy for force recruitment?

PubMed

Manuel, Marin; Heckman, C J

2011-10-19

Classical studies of the mammalian neuromuscular system have shown an impressive adaptation match between the intrinsic properties of motoneurons and the contractile properties of their motor units. In these studies, the rate at which motoneurons start to fire repetitively corresponds to the rate at which individual twitches start to sum, and the firing rate increases linearly with the amount of excitation ("primary range") up to the point where the motor unit develops its maximal force. This allows for the gradation of the force produced by a motor unit by rate modulation. In adult mouse motoneurons, however, we recently described a regime of firing ("subprimary range") that appears at lower excitation than what is required for the primary range, a finding that might challenge the classical conception. To investigate the force production of mouse motor units, we simultaneously recorded, for the first time, the motoneuron discharge elicited by intracellular ramps of current and the force developed by its motor unit. We showed that the motor unit developed nearly its maximal force during the subprimary range. This was found to be the case regardless of the input resistance of the motoneuron, the contraction speed, or the tetanic force of the motor unit. Our work suggests that force modulation in small mammals mainly relies on the number of motor units that are recruited rather than on rate modulation of individual motor units.

Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system.

PubMed Central

Yamamoto, O; Takakusa, N; Mishima, Y; Kominami, R; Muramatsu, M

1984-01-01

Sequences required for a faithful and efficient transcription of a cloned mouse ribosomal RNA gene (rDNA) are determined by testing a series of deletion mutants in an in vitro transcription system utilizing two kinds of mouse cellular extract. Deletion of sequences upstream of -40 or downstream of +52 causes only slight reduction in promoter activity as compared with the "wild-type" template. For upstream deletion mutants, the removal of a sequence between -40 and -35 causes a significant decrease in the capacity to direct efficient initiation. This decrease becomes more pronounced when the deletion reaches -32 and the sequence A-T-C-T-T-T, conserved among mouse, rat, and human rDNAs, is lost. Residual template activity is further reduced as more upstream sequence is deleted and finally becomes undetectable when the deletion is extended from -22 down to -17, corresponding to the loss of the conserved sequence T-A-T-T-G. As for downstream deletion mutants, the removal of the sequence downstream of +23 causes some (and further deletions up to +11 cause a more) serious decrease in template activity in vitro. These deletions involve other conserved sequences downstream of the transcription start site. However, the removal of the original transcription start site does not abolish the transcription initiation completely, provided that the whole upstream sequence is intact. Images PMID:6320178

Perception of Dialect Variation by Young Adults with High-Functioning Autism

ERIC Educational Resources Information Center

Clopper, Cynthia G.; Rohrbeck, Kristin L.; Wagner, Laura

2012-01-01

The linguistic profile of people with Autism spectrum disorders typically involves intact perceptual processing, accompanied by deficits in the social functions of language. In a series of three experiments, the impact of this profile on the perception of regional dialect was examined. Young adults with High-Functioning Autism exhibited similar…

Absence of Prenatal Forebrain Defects in the Dp(16)1Yey/+ Mouse Model of Down Syndrome

PubMed Central

Goodliffe, Joseph W.; Olmos-Serrano, Jose Luis; Aziz, Nadine M.; Pennings, Jeroen L.A.; Guedj, Faycal; Bianchi, Diana W.

2016-01-01

Studies in humans with Down syndrome (DS) show that alterations in fetal brain development are followed by postnatal deficits in neuronal numbers, synaptic plasticity, and cognitive and motor function. This same progression is replicated in several mouse models of DS. Dp(16)1Yey/+ (hereafter called Dp16) is a recently developed mouse model of DS in which the entire region of mouse chromosome 16 that is homologous to human chromosome 21 has been triplicated. As such, Dp16 mice may more closely reproduce neurodevelopmental changes occurring in humans with DS. Here, we present the first comprehensive cellular and behavioral study of the Dp16 forebrain from embryonic to adult stages. Unexpectedly, our results demonstrate that Dp16 mice do not have prenatal brain defects previously reported in human fetal neocortex and in the developing forebrains of other mouse models, including microcephaly, reduced neurogenesis, and abnormal cell proliferation. Nevertheless, we found impairments in postnatal developmental milestones, fewer inhibitory forebrain neurons, and deficits in motor and cognitive performance in Dp16 mice. Therefore, although this new model does not express prenatal morphological phenotypes associated with DS, abnormalities in the postnatal period appear sufficient to produce significant cognitive deficits in Dp16. SIGNIFICANCE STATEMENT Down syndrome (DS) leads to intellectual disability. Several mouse models have increased our understanding of the neuropathology of DS and are currently being used to test therapeutic strategies. A new mouse model that contains an expanded number of DS-related genes, known as Dp(16)1Yey/+ (Dp16), has been generated recently. We sought to determine whether the extended triplication creates a better phenocopy of DS-related brain pathologies. We measured embryonic development, forebrain maturation, and perinatal/adult behavior and revealed an absence of prenatal phenotypes in Dp16 fetal brain, but specific cellular and behavioral

Accuracy of a Mouse Bioassay for the Diagnosis of Botulism in Horses.

PubMed

Johnson, A L; McAdams-Gallagher, S C; Aceto, H

2016-07-01

The laboratory diagnosis of botulism in horses traditionally has relied upon the mouse bioassay (MBA). The accuracy of this test for the diagnosis of botulism in horses is unknown. Our goal was to determine the sensitivity, specificity, positive predictive value, and negative predictive value of the MBA on laboratory-processed fecal and gastrointestinal samples for foals and adult horses. Cases included all horses with a final clinical diagnosis of botulism that were admitted between 1986 and 2011 and had MBA testing performed. Controls included horses without botulism that were admitted during the same time period and had MBA testing performed. Retrospective study. Horses suspected of having botulism had fecal or (less commonly) gastrointestinal content samples tested using MBA. For every hospitalized botulism suspect, control samples were obtained from ≥1 additional hospitalized horses not suspected to have botulism. One hundred and twenty-nine adult horses and 253 adult controls were identified. Overall sensitivity of the MBA was only 32% but specificity was 97%. Forty-three foal cases and 21 foal controls were evaluated; sensitivity of the MBA was 53% and specificity was 100%. Positive predictive value was substantially higher (100% for foals and 89% for adults) than negative predictive value (51% for foals and 67% for adults). Mouse bioassay has low sensitivity but high specificity for the diagnosis of botulism in horses. Positive results are highly suggestive of botulism but negative results do not exclude the diagnosis. Unaffected horses and foals rarely shed C. botulinum in their feces. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Live imaging of mouse secondary palate fusion

PubMed Central

Kim, Seungil; Prochazka, Jan; Bush, Jeffrey O.

2017-01-01

LONG ABSTRACT The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled. PMID:28784960

Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

NASA Technical Reports Server (NTRS)

Barker, Ed; Abercromby, Kira J.; Abell, Paul

2009-01-01

A key objective of NASA s Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the 0.6 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectra is flat and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar panels with a strong band gap feature near 1 micron. The two spacecraft are spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but with fewer

Reflectance Spectra Comparison of Orbital Debris, Intact Spacecraft, and Intact Rocket Bodies in the GEO Regime

NASA Astrophysics Data System (ADS)

Albercromby, Kira J.; Abell, Paul; Barker, Ed

2009-03-01

A key objective of NASA's Orbital Debris program office at Johnson Space Center (JSC) is to characterize the debris environment by way of assessing the physical properties (type, mass, density, and size) of objects in orbit. Knowledge of the geosynchronous orbit (GEO) debris environment in particular can be used to determine the hazard probability at specific GEO altitudes and aid predictions of the future environment. To calculate an optical size from an intensity measurement of an object in the GEO regime, a 0.175 albedo is assumed currently. However, identification of specific material type or types could improve albedo accuracy and yield a more accurate size estimate for the debris piece. Using spectroscopy, it is possible to determine the surface materials of space objects. The study described herein used the NASA Infrared Telescope Facility (IRTF) to record spectral data in the ~ 0.65 to 2.5 micron regime on eight catalogued space objects. For comparison, all of the objects observed were in GEO or near-GEO. The eight objects consisted of two intact spacecraft, three rocket bodies, and three catalogued debris pieces. Two of the debris pieces stemmed from Titan 3C transtage breakup and the third is from COSMOS 2054. The reflectance spectra of the Titan 3C pieces share similar slopes (increasing with wavelength) and lack any strong absorption features. The COSMOS debris spectrum has a slight slope and has no absorption features. In contrast, the intact spacecraft show classic absorption features due to solar cells with a strong band gap feature near 1 micron. The two spacecraft were spin-stabilized objects and therefore have solar panels surrounding the outer surface. Two of the three rocket bodies are inertial upper stage (IUS) rocket bodies and have similar looking spectra. The slopes flatten out near 1.5 microns with absorption features in the near-infrared that are similar to that of white paint. The third rocket body has a similar flattening of slope but

Circadian clock-dependent and -independent posttranscriptional regulation underlies temporal mRNA accumulation in mouse liver

PubMed Central

Wang, Jingkui; Yeung, Jake; Gobet, Cédric; Sobel, Jonathan; Lück, Sarah; Molina, Nacho; Naef, Felix

2018-01-01

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1−/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver. PMID:29432155

Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

PubMed Central

Lovatt, Ditte; Ruble, Brittani K.; Lee, Jaehee; Dueck, Hannah; Kim, Tae Kyung; Fisher, Stephen; Francis, Chantal; Spaethling, Jennifer M.; Wolf, John A.; Grady, M. Sean; Ulyanova, Alexandra V.; Yeldell, Sean B.; Griepenburg, Julianne C.; Buckley, Peter T.; Kim, Junhyong; Sul, Jai-Yoon; Dmochowski, Ivan J.; Eberwine, James

2014-01-01

Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment. PMID:24412976

Young Children's Skill in Using a Mouse to Control a Graphical Computer Interface.

ERIC Educational Resources Information Center

Crook, Charles

1992-01-01

Describes a study that investigated the performance of preschoolers and children in the first three years of formal education on tasks that involved skills using a mouse-based control of a graphical computer interface. The children's performance is compared with that of novice adult users and expert users. (five references) (LRW)

Silica Aerogel Captures Cosmic Dust Intact

NASA Technical Reports Server (NTRS)

Tsou, P.

1994-01-01

The mesostructure of silica aerogel resembles stings of grapes, ranging in size from 10 to 100 angstrom. This fine mesostructure transmits nearly 90 percent of incident light in the visible, while providing sufficiently gentle dissipation of the kinetric energy of hypervelocity cosmic dust particles to permit their intact capture. We introduced silica aerogel in 1987 as capture medium to take advantage of its low density, fine mesostruicture and most importantly, its transparency, allowing optical location of captured micron sized particles.

Differential Effects of Focused and Unfocused Written Correction on the Accurate Use of Grammatical Forms by Adult ESL Learners

ERIC Educational Resources Information Center

Sheen, Younghee; Wright, David; Moldawa, Anna

2009-01-01

Building on Sheen's (2007) study of the effects of written corrective feedback (CF) on the acquisition of English articles, this article investigated whether direct focused CF, direct unfocused CF and writing practice alone produced differential effects on the accurate use of grammatical forms by adult ESL learners. Using six intact adult ESL…

Predominant Suppression of FSHβ-immunoreactivity after Long-Term Treatment of Intact and Castrate Adult Male Rats with the GnRH Agonist Deslorelin

PubMed Central

Smith, Arik W.; Asa, Cheryl S.; Edwards, Brian S.; Murdoch, William J.; Skinner, Donal C.

2017-01-01

GnRH agonists are used to treat gonadal steroid-dependent disorders in humans and contracept animals. These agonists are thought to work by desensitizing gonadotropes to GnRH, thereby suppressing FSH and LH secretion. It is not known whether changes occur in the cellular composition of the pituitary gland following chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0±0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labeled immunocytochemically for FSHβ and LHβ, or αGSU. Deslorelin suppressed testis weight by two thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotropes but the effect was specific to the FSHβ-ir cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotrope population. However, in the presence of testosterone, the proportion of gonadotropes that was FSHβ-ir increased in the remaining gonadotropes. There was no effect of treatment on the total LHβ-ir cell population although the loss of FSHβ in bi-hormonal cells increased the proportion of mono-hormonal LHβ-ir gonadotropes. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotrope population; thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotrope population per se is not eliminated by deslorelin but the ability of gonadotropes to synthesize FSHβ is compromised. We hypothesize that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male. PMID:22172059

Mouse Curve Biometrics

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

Schulz, Douglas A.

2007-10-08

A biometric system suitable for validating user identity using only mouse movements and no specialized equipment is presented. Mouse curves (mouse movements with little or no pause between them) are individually classied and used to develop classication histograms, which are representative of an individual's typical mouse use. These classication histograms can then be compared to validate identity. This classication approach is suitable for providing continuous identity validation during an entire user session.

Chemical composition and phytotoxicity of volatile essential oil from intact and fallen leaves of Eucalyptus citriodora.

PubMed

Batish, Daizy R; Singh, Harminder P; Setia, Nidhi; Kaur, Shalinder; Kohli, Ravinder K

2006-01-01

A total of 23 volatile constituents was identified and characterized by GC and GC-MS in the volatile essential oil extracted from intact (juvenile and adult) and fallen (senescent and leaf litter) leaves of lemon-scented eucalyptus (Eucalyptus citriodora Hook.). The leaves differed in their pigment, water and protein content, and C/N ratio. The oils were, in general, monoterpenoid in nature with 18 monoterpenes and 5 sesquiterpenes. However, a great variability in the amount of essential oils and their individual constituents was observed in different leaf tissues. The amount was maximum in the senescent leaves collected from the floor of the tree closely followed by that from juvenile leaves. In all, 19 constituents were identified in oil from juvenile and senescent leaves compared to 23 in adult leaves and 20 in leaf litter, respectively. Citronellal, a characteristic monoterpene of the oil reported hitherto was found to be more (77-78%) in the juvenile and senescent leaves compared to 48 and 54%, respectively, in the adult leaves and leaf litter. In the adult leaves, however, the content of citronellol--another important monoterpene-- was very high (21.9%) compared to other leaf types (7.8-12.2%). Essential oil and its two major monoterpenes viz. citronellal and citronellol were tested for their phytotoxicity against two weeds (Amaranthus viridis and Echinochloa crus-galli) and two crops (Triticum aestivum and Oryza sativa) under laboratory conditions. A difference in the phytotoxicity, measured in terms of seedling length and dry weight, of oil from different leaves and major monoterpenes was observed. Oil from adult leaves was found to be most phytotoxic although it occurs in smaller amount (on unit weight basis). The different toxicity of different oil types was due to the relative amount of individual monoterpenes present in the oil, their solubility and interactive action. The study concludes that oil from senescent and juvenile leaves being rich in

Increasing Adult Hippocampal Neurogenesis is Sufficient to Reduce Anxiety and Depression-Like Behaviors.

PubMed

Hill, Alexis S; Sahay, Amar; Hen, René

2015-09-01

Adult hippocampal neurogenesis is increased by antidepressants, and is required for some of their behavioral effects. However, it remains unclear whether expanding the population of adult-born neurons is sufficient to affect anxiety and depression-related behavior. Here, we use an inducible transgenic mouse model in which the pro-apoptotic gene Bax is deleted from neural stem cells and their progeny in the adult brain, and thereby increases adult neurogenesis. We find no effects on baseline anxiety and depression-related behavior; however, we find that increasing adult neurogenesis is sufficient to reduce anxiety and depression-related behaviors in mice treated chronically with corticosterone (CORT), a mouse model of stress. Thus, neurogenesis differentially affects behavior under baseline conditions and in a model of chronic stress. Moreover, we find no effect of increased adult hippocampal neurogenesis on hypothalamic-pituitary-adrenal (HPA) axis regulation, either at baseline or following chronic CORT administration, suggesting that increasing adult hippocampal neurogenesis can affect anxiety and depression-related behavior through a mechanism independent of the HPA axis. The use of future techniques to specifically inhibit BAX in the hippocampus could be used to augment adult neurogenesis, and may therefore represent a novel strategy to promote antidepressant-like behavioral effects.

Mouse Cleaning Apparatus and Method

NASA Technical Reports Server (NTRS)

Williams, Glenn L. (Inventor)

2005-01-01

The method of using the mouse pad cleaning apparatus is disclosed and claimed. The method comprises the steps of uncovering the mouse cleaning surface, applying the mouse and ball of the mouse to the cleaning surface, moving the mouse in a rotational pattern on the mouse cleaning surface, removing the mouse form the mouse cleaning surface, washing the cleaning surface, and covering the mouse cleaning surface. A mouse pad cleaning apparatus comprising a plurality of substrates, each said substrate having adhesive thereon, said plurality of substrates residing in and affixed to a receptacle. A single substrate having adhesive, which may be washable or non-washable, thereon may be employed. The washable adhesive may be an organopolysiloxane or gelatinous elastomer.

Distinct spatiotemporal expression of ISM1 during mouse and chick development.

PubMed

Osório, Liliana; Wu, Xuewei; Zhou, Zhongjun

2014-01-01

Isthmin 1 (ISM1) constitutes the founder of a new family of secreted proteins characterized by the presence of 2 functional domains: thrombospondin type 1 repeat (TSR1) and adhesion-associated domain in MUC4 and other proteins (AMOP). ISM1 was identified in the frog embryo as a member of the FGF8 synexpression group due to its expression in the brain midbrain-hindbrain boundary (MHB) or isthmus. In zebrafish, ISM1 was described as a WNT- and NODAL-regulated gene. The function of ISM1 remains largely elusive. So far, ISM1 has been described as an angiogenesis inhibitor that has a dual function in endothelial cell survival and cell death. For a better understanding of ISM1 function, we examined its spatiotemporal distribution in mouse and chick using RT-PCR, ISH, and IHC analyses. In the mouse, ISM1 transcripts are found in tissues such as the anterior mesendoderm, paraxial and lateral plate mesoderm, MHB and trunk neural tube, as well as in the somites and dermomyotome. In the newborn and adult, ISM1 is prominently expressed in the lung and brain. In addition to its putative role during embryonic and postnatal development, ISM1 may also be important for organ homeostasis in the adult. In the chick embryo, ISM1 transcripts are strongly detected in the ear, eye, and spinal cord primordia. Remarkable differences in ISM1 spatiotemporal expression were found during mouse and chick development, despite the high homology of ISM1 orthologs in these species.

Distinct spatiotemporal expression of ISM1 during mouse and chick development

PubMed Central

Osório, Liliana; Wu, Xuewei; Zhou, Zhongjun

2014-01-01

Isthmin 1 (ISM1) constitutes the founder of a new family of secreted proteins characterized by the presence of 2 functional domains: thrombospondin type 1 repeat (TSR1) and adhesion-associated domain in MUC4 and other proteins (AMOP). ISM1 was identified in the frog embryo as a member of the FGF8 synexpression group due to its expression in the brain midbrain–hindbrain boundary (MHB) or isthmus. In zebrafish, ISM1 was described as a WNT- and NODAL-regulated gene. The function of ISM1 remains largely elusive. So far, ISM1 has been described as an angiogenesis inhibitor that has a dual function in endothelial cell survival and cell death. For a better understanding of ISM1 function, we examined its spatiotemporal distribution in mouse and chick using RT-PCR, ISH, and IHC analyses. In the mouse, ISM1 transcripts are found in tissues such as the anterior mesendoderm, paraxial and lateral plate mesoderm, MHB and trunk neural tube, as well as in the somites and dermomyotome. In the newborn and adult, ISM1 is prominently expressed in the lung and brain. In addition to its putative role during embryonic and postnatal development, ISM1 may also be important for organ homeostasis in the adult. In the chick embryo, ISM1 transcripts are strongly detected in the ear, eye, and spinal cord primordia. Remarkable differences in ISM1 spatiotemporal expression were found during mouse and chick development, despite the high homology of ISM1 orthologs in these species. PMID:24675886

Aboveground Biomass Variability Across Intact and Degraded Forests in the Brazilian Amazon

NASA Technical Reports Server (NTRS)

Longo, Marcos; Keller, Michael; Dos-Santos, Maiza N.; Leitold, Veronika; Pinage, Ekena R.; Baccini, Alessandro; Saatchi, Sassan; Nogueira, Euler M.; Batistella, Mateus; Morton, Douglas C.

2016-01-01

Deforestation rates have declined in the Brazilian Amazon since 2005, yet degradation from logging, re, and fragmentation has continued in frontier forests. In this study we quantified the aboveground carbon density (ACD) in intact and degraded forests using the largest data set of integrated forest inventory plots (n 359) and airborne lidar data (18,000 ha) assembled to date for the Brazilian Amazon. We developed statistical models relating inventory ACD estimates to lidar metrics that explained70 of the variance across forest types. Airborne lidar-ACD estimates for intact forests ranged between 5.0 +/- 2.5 and 31.9 +/- 10.8 kg C m(exp -2). Degradation carbon losses were large and persistent. Sites that burned multiple times within a decade lost up to 15.0 +/- 0.7 kg C m(-2)(94%) of ACD. Forests that burned nearly15 years ago had between 4.1 +/- 0.5 and 6.8 +/- 0.3 kg C m(exp -2) (22-40%) less ACD than intact forests. Even for low-impact logging disturbances, ACD was between 0.7 +/- 0.3 and 4.4 +/- 0.4 kg C m(exp -2)(4-21%) lower than unlogged forests. Comparing biomass estimates from airborne lidar to existing biomass maps, we found that regional and pan-tropical products consistently overestimated ACD in degraded forests, under-estimated ACD in intact forests, and showed little sensitivity to res and logging. Fine-scale heterogeneity in ACD across intact and degraded forests highlights the benefits of airborne lidar for carbon mapping. Differences between airborne lidar and regional biomass maps underscore the need to improve and update biomass estimates for dynamic land use frontiers, to better characterize deforestation and degradation carbon emissions for regional carbon budgets and Reduce Emissions from Deforestation and forest Degradation(REDD+).

High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting.

PubMed

Ackermann, Amanda M; Zhang, Jia; Heller, Aryel; Briker, Anna; Kaestner, Klaus H

2017-03-01

α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER "knock-in" mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreER T2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. We utilized CRISPR-Cas9 technology to insert an IRES-CreER T2 sequence into the 3' UTR of the Glucagon ( Gcg ) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreER T2 mice. Recombination efficiency in GCG + pancreatic α-cells and glucagon-like peptide 1 positive (GLP1 + ) enteroendocrine L-cells was measured in Gcg-CreER T2 ; Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Tamoxifen injection of Gcg-CreER T2 ; Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreER T2 allele were phenotypically normal. We successfully derived a Gcg-CreER T2 mouse line that expresses CreER T2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing

Peptidomics Analysis of Transient Regeneration in the Neonatal Mouse Heart.

PubMed

Fan, Yi; Zhang, Qijun; Li, Hua; Cheng, Zijie; Li, Xing; Chen, Yumei; Shen, Yahui; Wang, Liansheng; Song, Guixian; Qian, Lingmei

2017-09-01

Neonatal mouse hearts have completely regenerative capability after birth, but the ability to regenerate rapidly lost after 7 days, the mechanism has not been clarified. Previous studies have shown that mRNA profile of adult mouse changed greatly compared to neonatal mouse. So far, there is no research of peptidomics related to heart regeneration. In order to explore the changes of proteins, enzymes, and peptides related to the transient regeneration, we used comparative petidomics technique to compare the endogenous peptides in the mouse heart of postnatal 1 and 7 days. In final, we identified 236 differentially expressed peptides, 169 of which were upregulated and 67 were downregulated in the postnatal 1 day heart, and also predicted 36 functional peptides associated with transient regeneration. The predicted 36 candidate peptides are located in the important domains of precursor proteins and/or contain the post-transcriptional modification (PTM) sites, which are involved in the biological processes of cardiac development, cardiac muscle disease, cell proliferation, necrosis, and apoptosis. In conclusion, for the first time, we compared the peptidomics profiles of neonatal heart between postnatal 1 day and postnatal 7 day. This study provides a new direction and an important basis for the mechanism research of transient regeneration in neonatal heart. J. Cell. Biochem. 118: 2828-2840, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Generation of a Slc39a8 hypomorph mouse: Markedly decreased ZIP8 Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} transporter expression

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

Wang, Bin; He, Lei; Dong, Hongbin

2011-07-01

Highlights: {yields} The mouse Slc39a8 gene encodes the ZIP8 transporter. {yields} ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter. {yields} A Slc39a8(neo/neo) hypomorph mouse, due to retention of the neo mini-gene, has been created. {yields} ZIP8 expression in utero is {approx}90% decreased in all tissues examined. {yields} This mouse model will be useful for studying developmental and in utero physiological functions of ZIP8. -- Abstract: Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter, moving bothmore » ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.« less

Mouse phenotyping.

PubMed

Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

2011-02-01

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

Characteristics of taurine release in slices from adult and developing mouse brain stem.

PubMed

Saransaari, P; Oja, S S

2006-07-01

Taurine has been thought to function as a regulator of neuronal activity, neuromodulator and osmoregulator. Moreover, it is essential for the development and survival of neural cells and protects them under cell-damaging conditions. Taurine is also involved in many vital functions regulated by the brain stem, including cardiovascular control and arterial blood pressure. The release of taurine has been studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release have not been systematically characterized in the brain stem. The properties of release of preloaded [(3)H]taurine were now characterized in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. In general, taurine release was found to be similar to that in other brain areas, consisting of both Ca(2+)-dependent and Ca(2+)-independent components. Moreover, the release was mediated by Na(+)-, Cl(-)-dependent transporters operating outwards, as both Na(+)-free and Cl(-) -free conditions greatly enhanced it. Cl(-) channel antagonists and a Cl(-) transport inhibitor reduced the release at both ages, indicating that a part of the release occurs through ion channels. Protein kinases appeared not to be involved in taurine release in the brain stem, since substances affecting the activity of protein kinase C or tyrosine kinase had no significant effects. The release was modulated by cAMP second messenger systems and phospholipases at both ages. Furthermore, the metabotropic glutamate receptor agonists likewise suppressed the K(+)-stimulated release at both ages. In the immature brain stem, the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) potentiated taurine release in a receptor-mediated manner. This could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse.

PubMed

Baudouy, Delphine; Michiels, Jean-François; Vukolic, Ana; Wagner, Kay-Dietrich; Wagner, Nicole

2017-10-26

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Uncompensated polyuria in a mouse model of Bartter's syndrome

PubMed Central

Takahashi, Nobuyuki; Chernavvsky, Daniel R.; Gomez, R. Ariel; Igarashi, Peter; Gitelman, Hillel J.; Smithies, Oliver

2000-01-01

We have used homologous recombination to disrupt the mouse gene coding for the NaK2Cl cotransporter (NKCC2) expressed in kidney epithelial cells of the thick ascending limb and macula densa. This gene is one of several that when mutated causes Bartter's syndrome in humans, a syndrome characterized by severe polyuria and electrolyte imbalance. Homozygous NKCC2−/− pups were born in expected numbers and appeared normal. However, by day 1 they showed signs of extracellular volume depletion (hematocrit 51%; wild type 37%). They subsequently failed to thrive. By day 7, they were small and markedly dehydrated and exhibited renal insufficiency, high plasma potassium, metabolic acidosis, hydronephrosis of varying severity, and high plasma renin concentrations. None survived to weaning. Treatment of −/− pups with indomethacin from day 1 prevented growth retardation and 10% treated for 3 weeks survived, although as adults they exhibited severe polyuria (10 ml/day), extreme hydronephrosis, low plasma potassium, high blood pH, hypercalciuria, and proteinuria. Wild-type mice treated with furosemide, an inhibitor of NaK2Cl cotransporters, have a phenotype similar to the indomethacin-rescued −/− adults except that hydronephrosis was mild. The polyuria, hypercalciuria, and proteinuria of the −/− adults and furosemide-treated wild-type mice were unresponsive to inhibitors of the renin angiotensin system, vasopressin, and further indomethacin. Thus absence of NKCC2 in the mouse causes polyuria that is not compensated elsewhere in the nephron. The NKCC2 mutant animals should be valuable for uncovering new pathophysiologic and therapeutic aspects of genetic disturbances in water and electrolyte recovery by the kidney. PMID:10779555

The future of criminal violence: juveniles tried as adults.

PubMed

Kirkish, P; Sreenivasan, S; Welsh, R; Van Gorp, W; Eth, S; Shoptaw, S; Ling, W

2000-01-01

Juveniles tried as adults (JTA) represent a select and small subsample of juvenile offenders. This study seeks to provide a profile of habitually violent JTAs transferred to the adult penal system and to compare them with their adult counterparts. Twenty-nine incarcerated violent male juveniles tried as adults were compared with a sample of 27 incarcerated violent male offenders across demographic, neuropsychological, criminal history, psychopathy, and substance abuse variables. The JTAs were characterized by a high rate of gang membership (96%), substance abuse (alcohol, marijuana, and phenylcyclidene), and use of guns. In the juvenile sample, 65 percent used guns in violence not leading to arrest, and 93 percent used guns in a violent crime leading to arrest. Juvenile offenders were similar to their adult counterparts in patterns of criminality, although adult offenders had higher psychopathy scores. Both groups revealed generally intact neuropsychological functioning with the exception of a higher rate of perseverative responses in the adult sample. The results are discussed in terms of the implication of the degree of violence in a young offender population.

Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain.

PubMed

Kosi, Nina; Alić, Ivan; Kolačević, Matea; Vrsaljko, Nina; Jovanov Milošević, Nataša; Sobol, Margarita; Philimonenko, Anatoly; Hozák, Pavel; Gajović, Srećko; Pochet, Roland; Mitrečić, Dinko

2015-02-09

The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Sympathetic Nervous Regulation of Calcium and Action Potential Alternans in the Intact Heart.

PubMed

Winter, James; Bishop, Martin J; Wilder, Catherine D E; O'Shea, Christopher; Pavlovic, Davor; Shattock, Michael J

2018-01-01

Rationale: Arrhythmogenic cardiac alternans are thought to be an important determinant for the initiation of ventricular fibrillation. There is limited information on the effects of sympathetic nerve stimulation (SNS) on alternans in the intact heart and the conclusions of existing studies, focused on investigating electrical alternans, are conflicted. Meanwhile, several lines of evidence implicate instabilities in Ca handling, not electrical restitution, as the primary mechanism underpinning alternans. Despite this, there have been no studies on Ca alternans and SNS in the intact heart. The present study sought to address this, by application of voltage and Ca optical mapping for the simultaneous study of APD and Ca alternans in the intact guinea pig heart during direct SNS. Objective : To determine the effects of SNS on APD and Ca alternans in the intact guinea pig heart and to examine the mechanism(s) by which the effects of SNS are mediated. Methods and Results : Studies utilized simultaneous voltage and Ca optical mapping in isolated guinea pig hearts with intact innervation. Alternans were induced using a rapid dynamic pacing protocol. SNS was associated with rate-independent shortening of action potential duration (APD) and the suppression of APD and Ca alternans, as indicated by a shift in the alternans threshold to faster pacing rates. Qualitatively similar results were observed with exogenous noradrenaline perfusion. In contrast with previous reports, both SNS and noradrenaline acted to flatten the slope of the electrical restitution curve. Pharmacological block of the slow delayed rectifying potassium current (I Ks ), sufficient to abolish I Ks -mediated APD-adaptation, partially reversed the effects of SNS on pacing-induced alternans. Treatment with cyclopiazonic acid, an inhibitor of the sarco(endo)plasmic reticulum ATPase, had opposite effects to that of SNS, acting to increase susceptibility to alternans, and suggesting that accelerated Ca reuptake

Maternal high-protein diet during pregnancy, but not during suckling, induced altered expression of an increasing number of hepatic genes in adult mouse offspring.

PubMed

Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Metges, Cornelia C

2016-04-01

Indirect effects of a high-protein maternal diet are not well understood. In this study, we analyzed short-term and sustainable effects of a prenatal versus early postnatal maternal high-protein diet on growth and hepatic gene expression in mouse offspring. Dams were exposed to an isoenergetic high-protein (HP, 40 % w/w) diet during pregnancy or lactation. Growth and hepatic expression profiles of male offspring were evaluated directly after weaning and 150 days after birth. Offspring from two dietary groups, high-protein diet during pregnancy and control diet during lactation (HPC), and control diet during pregnancy and high-protein diet during lactation (CHP), were compared with offspring (CC) from control-fed dams. Maternal CHP treatment was associated with sustained offspring growth retardation, but decreased numbers of affected hepatic genes in adults compared to weanlings. In contrast, offspring of the HPC group did not show persistent effects on growth parameters, but the number of affected hepatic genes was even increased at adult age. In both dietary groups, however, only a small subset of genes was affected in weanlings as well as in adults. We conclude that (1) prenatal and early postnatal maternal HP diet caused persistent, but (2) different effects and partially complementary trends on growth characteristics and on the hepatic transcriptome and associated pathways and that (3) only a small number of genes and associated upstream regulators might be involved in passing early diet-induced imprints to adulthood.

Morphology of isolated mouse inner cell masses developing in vitro

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

Wiley, L.M.; Spindle, A.I.; Pedersen, R.A.

1978-01-01

The purpose of this study was to examine the developmental capacity of the mouse inner cell mass (ICM) in the absence of the trophoblast. ICMs were isolated from blastocysts by immunosurgery and cultured under conditions that support egg cylinder formation by intact blastocysts. After 2 or 3 days of culture, the ICMs consisted of an outer layer of endoderm and an inner layer of ectoderm that had cavitated centrally. By 4 or 5 days of culture, 25 to 60% of these ICMs had developed into paired cysts, apparently by secondary cavity formation. The inner cell layer surrounding this secondary cavitymore » resembled the extraembryonic ectoderm of cultured egg cylinders. By 6 days of culture, 60% of the ICMs had expanded into yolk sac-like structures that subsequently produced capillaries containing blood cells. The ICMs appeared to develop mesoderm in two distinct ways. A few of them developed mesoderm as a third layer of cells in the cleft separating endoderm and ectoderm, presumably by migrating from the inner, ectodermal layer, through the primitive streak, as in the intact egg cylinder. In the rest of the ICMs the embryonic ectoderm gradually differentiated into mesoderm while still in the inner layer, without primitive streak formation. We suggest, therefore, that the continuous presence of the trophoblast or of its derivatives is not required for the cytodifferentiation of mesoderm although it may be important in establishing embryonic polarity or in providinginductive signals necessary for the morphogenetic aspects of mesoderm differentiation, specifically primitive streak formation.« less

Identification of the human homolog of the imprinted mouse Air non-coding RNA

PubMed Central

Yotova, Iveta Y.; Vlatkovic, Irena M.; Pauler, Florian M.; Warczok, Katarzyna E.; Ambros, Peter F.; Oshimura, Mitsuo; Theussl, Hans-Christian; Gessler, Manfred; Wagner, Erwin F.; Barlow, Denise P.

2010-01-01

Genomic imprinting is widely conserved amongst placental mammals. Imprinted expression of IGF2R, however, differs between mice and humans. In mice, Igf2r imprinted expression is seen in all fetal and adult tissues. In humans, adult tissues lack IGF2R imprinted expression, but it is found in fetal tissues and Wilms' tumors where it is polymorphic and only seen in a small proportion of tested samples. Mouse Igf2r imprinted expression is controlled by the Air (Airn) ncRNA whose promoter lies in an intronic maternally-methylated CpG island. The human IGF2R gene carries a homologous intronic maternally-methylated CpG island of unknown function. Here, we use transfection and transgenic studies to show that the human IGF2R intronic CpG island is a ncRNA promoter. We also identify the same ncRNA at the endogenous human locus in 16–40% of Wilms' tumors. Thus, the human IGF2R gene shows evolutionary conservation of key features that control imprinted expression in the mouse. PMID:18789384

Predictors of attachment security in preschool children from intact and divorced families.

PubMed

Nair, Hira; Murray, Ann D

2005-09-01

The authors selected 58 mother-child dyads from divorced and intact families to participate in a study on the impact of divorce on preschoolers' attachment security. The authors explored pathways that lead to security of attachment. They found that mothers from divorced families were younger, had lower income levels, and had lower levels of education compared with their intact counterparts. Divorced mothers also reported significantly higher levels of stress, depression, need for social support, and conflict with their spouses. Mothers from intact families were more likely to use positive (authoritative) parenting styles compared with divorced mothers. Children in the divorced group had lower security scores on the Attachment Q-Set instrument (E. Waters, 1995). Regression analyses indicated that parenting style made a direct (independent) contribution to attachment security. In addition, temperament was related to attachment security, but temperament did not diminish the association of parenting style with attachment security. Furthermore, regression analyses indicated that the relationship of divorce to attachment security was mediated by parenting style.

Multitasking in older adults with type 2 diabetes: A cross-sectional analysis.

PubMed

Rucker, Jason L; McDowd, Joan M; Mahnken, Jonathan D; Burns, Jeffrey M; Sabus, Carla H; Britton-Carpenter, Amanda J; Utech, Nora B; Kluding, Patricia M

2017-01-01

Deficits in the ability to multitask contribute to gait abnormalities and falls in many at-risk populations. However, it is unclear whether older adults with type 2 diabetes mellitus (DM) also demonstrate impairments in multitasking. The purpose of this study was to compare multitasking performance in cognitively intact older adults with and without DM and explore its relationship to measures of gait and functional ability. We performed a cross-sectional analysis of 40 individuals aged 60 and older with type 2 DM and a matched group of 40 cognitively intact older adults without DM. Multitasking was examined via the ambulatory Walking and Remembering Test (WART) and seated Pursuit Rotor Test (PRT). Self-selected normal and fast walking speed and stride length variability were quantitatively measured, and self-reported functional ability was assessed via the Late Life Function and Disability Index (LLFDI). Participants with DM walked slower and took more steps off path when multitasking during the WART. No between-group differences in multitasking performance were observed on the PRT. Multitasking performance demonstrated little correlation with gait and functional ability in either group. Older adults with DM appear to perform poorly on an ambulatory measure of multitasking. However, we analyzed a relatively small, homogenous sample of older adults with and without type 2 DM and factors such as peripheral neuropathy and the use of multiple comparisons complicate interpretation of the data. Future research should explore the interactions between multitasking and safety, fall risk, and function in this vulnerable population. Clinicians should recognize that an array of factors may contribute to gait and physical dysfunction in older adults with type 2 diabetes, and be prepared to assess and intervene appropriately.

Toward best practices in data processing and analysis for intact biotherapeutics by MS in quantitative bioanalysis.

PubMed

Kellie, John F; Kehler, Jonathan R; Karlinsey, Molly Z; Summerfield, Scott G

2017-12-01

Typically, quantitation of biotherapeutics from biological matrices by LC-MS is based on a surrogate peptide approach to determine molecule concentration. Recent efforts have focused on quantitation of the intact protein molecules or larger mass subunits of monoclonal antibodies. To date, there has been limited guidance for large or intact protein mass quantitation for quantitative bioanalysis. Intact- and subunit-level analyses of biotherapeutics from biological matrices are performed at 12-25 kDa mass range with quantitation data presented. Linearity, bias and other metrics are presented along with recommendations made on the viability of existing quantitation approaches. This communication is intended to start a discussion around intact protein data analysis and processing, recognizing that other published contributions will be required.

SOLITARY CHEMORECEPTOR CELL SURVIVAL IS INDEPENDENT OF INTACT TRIGEMINAL INNERVATION

PubMed Central

Gulbransen, Brian; Silver, Wayne; Finger, Tom

2008-01-01

Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al., 2003). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al., 2003) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. PMID:18300260

[Comparative ultrastructural study of parotid gland, lacrimal gland and pituitary gland between miniature pig and mouse].

PubMed

Yan, Xing; Hai, Bo; Sun, Yi-lin; Zhang, Chun-mei; Wang, Song-ling

2009-02-01

To study the ultrastructure of parotid glands, lacrimal glands and pituitary glands between miniature pig and mouse. Five adult miniature pigs and 5 mice were studied. Ultrastructure of their parotid glands, lacrimal glands, and pituitary glands was observed. The secretary granules in acinar cell of miniature pig parotid glands showed higher density and more aequalis than those of mice. The cell apparatus in acinar cell of mouse parotid glands were more plentiful than those of miniature pigs. The secretary granules on blood vessel wall were richer in parotid gland of miniature pigs compared with mouse parotid gland. Lacrimal gland had the similar ultrastructure to parotid gland in these two animals. Many blood vessel antrum were found in pituitary glands of these two animals. Compared with mouse parotid glands, there are more secretary granules in acinar cells and vascular endothelial cells in miniature pig parotid glands, which might enter blood stream and have function of endocrine secretion.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E*

PubMed Central

Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit

2017-01-01

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. PMID:27920204

Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

PubMed

Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

2014-07-23

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

Global forest loss disproportionately erodes biodiversity in intact landscapes.

PubMed

Betts, Matthew G; Wolf, Christopher; Ripple, William J; Phalan, Ben; Millers, Kimberley A; Duarte, Adam; Butchart, Stuart H M; Levi, Taal

2017-07-27

Global biodiversity loss is a critical environmental crisis, yet the lack of spatial data on biodiversity threats has hindered conservation strategies. Theory predicts that abrupt biodiversity declines are most likely to occur when habitat availability is reduced to very low levels in the landscape (10-30%). Alternatively, recent evidence indicates that biodiversity is best conserved by minimizing human intrusion into intact and relatively unfragmented landscapes. Here we use recently available forest loss data to test deforestation effects on International Union for Conservation of Nature Red List categories of extinction risk for 19,432 vertebrate species worldwide. As expected, deforestation substantially increased the odds of a species being listed as threatened, undergoing recent upgrading to a higher threat category and exhibiting declining populations. More importantly, we show that these risks were disproportionately high in relatively intact landscapes; even minimal deforestation has had severe consequences for vertebrate biodiversity. We found little support for the alternative hypothesis that forest loss is most detrimental in already fragmented landscapes. Spatial analysis revealed high-risk hot spots in Borneo, the central Amazon and the Congo Basin. In these regions, our model predicts that 121-219 species will become threatened under current rates of forest loss over the next 30 years. Given that only 17.9% of these high-risk areas are formally protected and only 8.9% have strict protection, new large-scale conservation efforts to protect intact forests are necessary to slow deforestation rates and to avert a new wave of global extinctions.

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

PubMed Central

Kocurek, Klaudia I.; Griffiths, Rian L.

2018-01-01

Abstract Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed. PMID:29607564

Effect of exercise and exogenous glucocorticoid on serum level of intact parathyroid hormone.

PubMed

Tsai, K S; Lin, J C; Chen, C K; Cheng, W C; Yang, C H

1997-11-01

Most previous studies suggest that physical exercise, or physiological response to exercise such as cortisol and adrenaline secretion regulate parathyroid hormone (PTH) secretion in humans. To investigate the effects and possible interaction of exercise and excessive glucocorticoid on PTH secretion, we examined the serum of levels of intact-PTH, cortisol, adrenocorticotrophic hormone (ACTH), calcium, magnesium and phosphorus before and during one-hour of bicycle-ergometric exercise at 60% of maximal oxygen uptake. These exercise tests were performed on eight Chinese male volunteers aged between 20 and 25 years, once with and once without pretreatment with 0.5 mg of dexamethasone taken orally 9.5 hours in advance. The results showed that dexamethasone pretreatment significantly lowered basal levels of cortisol and ACTH, but intact PTH did not change. After 60 minutes of bicycling, intact PTH level increases by 50% of baseline both with and without dexamethasone pretreatment. Serum levels of calcium, corrected for changes in serum albumin concentration, phosphorus and magnesium also increased in both cases. This study demonstrated an increase of intact-PTH with exercise which was not associated with hypocalcemia or hypomagnesemia, and was not altered in the presence of mild exogenous glucocorticoid excess and suppressed endogenous cortisol secretion.

Mouse TCOF1 is expressed widely, has motifs conserved in nucleolar phosphoproteins, and maps to chromosome 18.

PubMed

Paznekas, W A; Zhang, N; Gridley, T; Jabs, E W

1997-09-08

Mutations in the human TCOF1 gene have been identified in patients with Treacher Collins Syndrome (Mandibulofacial Dysostosis), an autosomal dominant condition affecting the craniofacial region. We report the isolation of the entire mouse Tcof1 coding sequence (3960 bp) by performing a computer-based search for mouse cDNA clones homologous to TCOF1 and generating overlapping RT-PCR products from mouse RNA. Tcof1 is a 1320 amino acid protein of 135 kd with 61.4% identity to TCOF1 and displays repeating motifs enriched for serine- and acidic amino acid-rich regions with potential phosphorylation sites and putative nuclear localization signals. Tcof1 maps to the mouse chromosome 18 region syntenic with human chromosome 5q32-->q33 which contains the TCOF1 locus. Northern blot hybridization indicates Tcof1 expression is ubiquitous in adult tissues and in the embryonic stage, is elevated at 11 dpc when the branchial arches and facial swellings are present in mouse. Our results are consistent with TCOF1 mutations leading to the Treacher Collins syndrome phenotype.

Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.

PubMed

Okubo, Tadashi; Clark, Cheryl; Hogan, Brigid L M

2009-02-01

The epithelium of the mouse tongue and soft palate consists of at least three distinct epithelial cell populations: basal cells, keratinized cells organized into filiform and fungiform papillae, and taste receptor cells present in tight clusters known as taste buds in the fungiform and circumvallate papillae and soft palate. All three cell types develop from the simple epithelium of the embryonic tongue and palate, and are continually replaced in the adult by cell turnover. Previous studies using pulse-chase tritiated thymidine labeling in the adult mouse provided evidence for a high rate of cell turnover in the keratinocytes (5-7 days) and taste buds (10 days). However, little is known about the localization and phenotype of the long-term stem or progenitor cells that give rise to the mature taste bud cells and surrounding keratinocytes in these gustatory tissues. Here, we make use of a tamoxifen-inducible K14-CreER transgene and the ROSA26 LacZ reporter allele to lineage trace the mature keratinocytes and taste bud cells of the early postnatal and adult mouse tongue and soft palate. Our results support the hypothesis that both the pore keratinocytes and receptor cells of the taste bud are derived from a common K14(+)K5(+)Trp63(+)Sox2(+) population of bipotential progenitor cells located outside the taste bud. The results are also compatible with models in which the keratinocytes of the filiform and fungiform papillae are derived from basal progenitor cells localized at the base of these structures.

Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains.

PubMed

Müllenbroich, M Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel Van't; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S

2015-10-01

Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains.

Effect of Male House Mouse Pheromone Components on Behavioral Responses of Mice in Laboratory and Field Experiments.

PubMed

Musso, Antonia E; Gries, Regine; Zhai, Huimin; Takács, Stephen; Gries, Gerhard

2017-03-01

Urine of male house mice, Mus musculus, is known to have primer pheromone effects on the reproductive physiology of female mice. Urine-mediated releaser pheromone effects that trigger certain behavioral responses are much less understood, and no field studies have investigated whether urine deposits by male or female mice, or synthetic mouse pheromone, increase trap captures of mice. In field experiments, we baited traps with bedding soiled with urine and feces of caged female or male mice, and recorded captures of mice in these and in control traps containing clean bedding. Traps baited with female bedding preferentially captured adult males, whereas traps baited with male bedding preferentially captured juvenile and adult females, indicating the presence of male- and female-specific sex pheromones in soiled bedding. Analyses of headspace volatiles emanating from soiled bedding by gas chromatography/mass spectrometry revealed that 3,4-dehydro-exo-brevicomin (DEB) was seven times more prevalent in male bedding and that 2-sec-butyl-4,5-dihydrothiazole (DHT) was male-specific. In a follow-up field experiment, traps baited with DEB and DHT captured 4 times more female mice than corresponding control traps, thus indicating that DEB and DHT are sex attractant pheromone components of house mouse males. Our study provides impetus to identify the sex attractant pheromone of female mice, and to develop synthetic mouse pheromone as a lure to enhance the efficacy of trapping programs for mouse control.

Vesicular monoamine transporter-1 (VMAT-1) mRNA and immunoreactive proteins in mouse brain.

PubMed

Ashe, Karen M; Chiu, Wan-Ling; Khalifa, Ahmed M; Nicolas, Antoine N; Brown, Bonnie L; De Martino, Randall R; Alexander, Clayton P; Waggener, Christopher T; Fischer-Stenger, Krista; Stewart, Jennifer K

2011-01-01

Vesicular monoamine transporter 1 (VMAT-1) mRNA and protein were examined (1) to determine whether adult mouse brain expresses full-length VMAT-1 mRNA that can be translated to functional transporter protein and (2) to compare immunoreactive VMAT-1 proteins in brain and adrenal. VMAT-1 mRNA was detected in mouse brain with RT-PCR. The cDNA was sequenced, cloned into an expression vector, transfected into COS-1 cells, and cell protein was assayed for VMAT-1 activity. Immunoreactive proteins were examined on western blots probed with four different antibodies to VMAT-1. Sequencing confirmed identity of the entire coding sequences of VMAT-1 cDNA from mouse medulla oblongata/pons and adrenal to a Gen-Bank reference sequence. Transfection of the brain cDNA into COS-1 cells resulted in transporter activity that was blocked by the VMAT inhibitor reserpine and a proton ionophore, but not by tetrabenazine, which has a high affinity for VMAT-2. Antibodies to either the C- or N- terminus of VMAT-1 detected two proteins (73 and 55 kD) in transfected COS-1 cells. The C-terminal antibodies detected both proteins in extracts of mouse medulla/pons, cortex, hypothalamus, and cerebellum but only the 73 kD protein and higher molecular weight immunoreactive proteins in mouse adrenal and rat PC12 cells, which are positive controls for rodent VMAT-1. These findings demonstrate that a functional VMAT-1 mRNA coding sequence is expressed in mouse brain and suggest processing of VMAT-1 protein differs in mouse adrenal and brain.

Characterization of subtle brain abnormalities in a mouse model of Hedgehog pathway antagonist-induced cleft lip and palate.

PubMed

Lipinski, Robert J; Holloway, Hunter T; O'Leary-Moore, Shonagh K; Ament, Jacob J; Pecevich, Stephen J; Cofer, Gary P; Budin, Francois; Everson, Joshua L; Johnson, G Allan; Sulik, Kathleen K

2014-01-01

Subtle behavioral and cognitive deficits have been documented in patient cohorts with orofacial clefts (OFCs). Recent neuroimaging studies argue that these traits are associated with structural brain abnormalities but have been limited to adolescent and adult populations where brain plasticity during infancy and childhood may be a confounding factor. Here, we employed high resolution magnetic resonance microscopy to examine primary brain morphology in a mouse model of OFCs. Transient in utero exposure to the Hedgehog (Hh) signaling pathway antagonist cyclopamine resulted in a spectrum of facial dysmorphology, including unilateral and bilateral cleft lip and palate, cleft of the secondary palate only, and a non-cleft phenotype marked by midfacial hypoplasia. Relative to controls, cyclopamine-exposed fetuses exhibited volumetric differences in several brain regions, including hypoplasia of the pituitary gland and olfactory bulbs, hyperplasia of the forebrain septal region, and expansion of the third ventricle. However, in affected fetuses the corpus callosum was intact and normal division of the forebrain was observed. This argues that temporally-specific Hh signaling perturbation can result in typical appearing OFCs in the absence of holoprosencephaly--a condition classically associated with Hh pathway inhibition and frequently co-occurring with OFCs. Supporting the premise that some forms of OFCs co-occur with subtle brain malformations, these results provide a possible ontological basis for traits identified in clinical populations. They also argue in favor of future investigations into genetic and/or environmental modulation of the Hh pathway in the etiopathogenesis of orofacial clefting.

Dor procedure for pulmonary atresia with intact ventricular septum in an infant.

PubMed

Kang, Yoonjin; Kwak, Jae Gun; Kim, Eung Rae; Kim, Woong-Han

2018-02-01

Pulmonary atresia with intact ventricular septum and right ventricle-dependent coronary circulation confers a high risk for myocardial infarction and complications of ischaemia, including left ventricle aneurysms. We describe an infant with pulmonary atresia with intact ventricular septum and right ventricle-dependent coronary circulation and a left ventricle apical aneurysm who successfully underwent the Dor procedure and a subsequent Fontan operation. Surgery for postinfarction left ventricle aneurysm can be considered in infants. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.

PubMed

Choi, Hannah; Zhang, Lei; Cembrowski, Mark S; Sabottke, Carl F; Markowitz, Alexander L; Butts, Daniel A; Kath, William L; Singer, Joshua H; Riecke, Hermann

2014-09-15

In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. Copyright © 2014 the American Physiological Society.

Kinetochore identification in micronuclei in mouse bone-marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents.

PubMed

Gudi, R; Sandhu, S S; Athwal, R S

1990-10-01

An in vivo micronucleus assay using mouse bone marrow for identifying the ability of chemicals to induce aneuploidy and/or chromosome breaks is described. Micronucleus formation in bone-marrow erythrocytes of mice is commonly used as an index for evaluating the clastogenicity of environmental agents. However, micronuclei may also originate from intact lagging chromosomes resulting from the effect of aneuploidy-inducing agents. We have used immunofluorescent staining using anti-kinetochore antibodies to classify micronuclei for the presence or absence of kinetochores. Micronuclei positive for kinetochores are assumed to contain intact chromosomes and result from induced aneuploidy; while those negative for kinetochores contain acentric chromosomal fragments and originate from clastogenic events. The assay was evaluated using X-irradiation (a known clastogen) and vincristine sulfate (an aneuploidy-inducing agent). A dose-related response for the induction of micronuclei was observed for both agents. Micronuclei induced by X-irradiation were negative for kinetochores while the majority of the micronuclei resulting from vincristine treatment contained kinetochores. Thus, the micronucleus assay in combination with immunofluorescent staining for kinetochores may provide a useful method to simultaneously assess the ability of chemicals to induce aneuploidy and/or chromosome breaks.

Gender differences in performance of script analysis by older adults.

PubMed

Helmes, E; Bush, J D; Pike, D L; Drake, D G

2006-12-01

Script analysis as a test of executive functions is presumed sensitive to cognitive changes seen with increasing age. Two studies evaluated if gender differences exist in performance on scripts for familiar and unfamiliar tasks in groups of cognitively intact older adults. In Study 1, 26 older adults completed male and female stereotypical scripts. Results were not significant but a tendency was present, with genders making fewer impossible errors on the gender-typical script. Such an interaction was also noted in Study 2, which contrasted 50 older with 50 younger adults on three scripts, including a script with neutral familiarity. The pattern of significant interactions for errors suggested the need to use scripts that are based upon tasks that are equally familiar to both genders.

Solitary chemoreceptor cell survival is independent of intact trigeminal innervation.

PubMed

Gulbransen, Brian; Silver, Wayne; Finger, Thomas E

2008-05-01

Nasal solitary chemoreceptor cells (SCCs) are a population of specialized chemosensory epithelial cells presumed to broaden trigeminal chemoreceptivity in mammals (Finger et al. [2003] Proc Natl Acad Sci USA 100:8981-8986). SCCs are innervated by peptidergic trigeminal nerve fibers (Finger et al. [2003]) but it is currently unknown if intact innervation is necessary for SCC development or survival. We tested the dependence of SCCs on innervation by eliminating trigeminal nerve fibers during development with neurogenin-1 knockout mice, during early postnatal development with capsaicin desensitization, and during adulthood with trigeminal lesioning. Our results demonstrate that elimination of innervation at any of these times does not result in decreased SCC numbers. In conclusion, neither SCC development nor mature cell maintenance is dependent on intact trigeminal innervation. (c) 2008 Wiley-Liss, Inc.

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

PubMed

Sun, Chengsan; Hummler, Edith; Hill, David L

2017-01-18

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role

Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract

PubMed Central

Sun, Chengsan; Hummler, Edith

2017-01-01

Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent “pruning” of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. SIGNIFICANCE STATEMENT Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case

Visual Speech Fills in Both Discrimination and Identification of Non-Intact Auditory Speech in Children

ERIC Educational Resources Information Center

Jerger, Susan; Damian, Markus F.; McAlpine, Rachel P.; Abdi, Herve

2018-01-01

To communicate, children must discriminate and identify speech sounds. Because visual speech plays an important role in this process, we explored how visual speech influences phoneme discrimination and identification by children. Critical items had intact visual speech (e.g. baez) coupled to non-intact (excised onsets) auditory speech (signified…

Ipsilateral intact fibula as a predictor of tibial plafond fracture pattern and severity.

PubMed

Luk, Pamela C; Charlton, Timothy P; Lee, Jackson; Thordarson, David B

2013-10-01

The objective of this study was to determine whether there is a difference in fracture pattern and severity of comminution between tibial plafond fractures with and without associated fibular fractures using computed tomography (CT). We hypothesized that the presence of an intact fibula was predictive of increased tibial plafond fracture severity. This was a case control, radiographic review performed at a single level I university trauma center. Between November 2007 and July 2011, 104 patients with 107 operatively treated tibial pilon fractures and preoperative CT scans were identified: 70 patients with 71 tibial plafond fractures had associated fibular fractures, and 34 patients with 36 tibial plafond fractures had intact fibulas. Four criteria were compared between the 2 groups: AO/OTA classification of distal tibia fractures, Topliss coronal and sagittal fracture pattern classification, plafond region of greatest comminution, and degree of proximal extension of fracture line. The intact fibula group had greater percentages of AO/OTA classification B2 type (5.5 vs 0, P = .046) and B3 type (52.8 vs 28.2, P = .013). Conversely, the percentage of AO/OTA classification C3 type was greater in the fractured fibula group (53.5 vs 30.6, P = .025). Evaluation using the Topliss sagittal and coronal classifications revealed no difference between the 2 groups (P = .226). Central and lateral regions of the plafond were the most common areas of comminution in fractured fibula pilons (32% and 31%, respectively). The lateral region of the plafond was the most common area of comminution in intact fibula pilon fractures (42%). There was no statistically significant difference (P = .71) in degree of proximal extension of fracture line between the 2 groups. Tibial plafond fractures with intact fibulas were more commonly associated with AO/OTA classification B-type patterns, whereas those with fractured fibulas were more commonly associated with C-type patterns. An intact fibula

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

PubMed

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3-independent mechanism.

PubMed

Gwynn, B; Ciciotte, S L; Hunter, S J; Washburn, L L; Smith, R S; Andersen, S G; Swank, R T; Dell'Angelica, E C; Bonifacino, J S; Eicher, E M; Peters, L L

2000-12-15

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)

Comparative Evaluation of Impact Strength of Fragment Bonded Teeth and Intact Teeth: An In Vitro Study

PubMed Central

Venugopal, L; Lakshmi, M Narasimha; Babu, Devatha Ashok; Kiran, V Ravi

2014-01-01

Background: To test and compare the impact strength of fragment bonded teeth with that of intact teeth by using impact testing machine (pendulum type) as a mode of load. Materials and Methods: Forty extracted, maxillary, central incisors selected for this study (20 control group and 20 experimental group). In experimental group, teeth crowns were fractured with a microtome at 2.5 mm from mesioincisal angle cervically, fractured portion is attached to original crown portion with 3 M single bond dentin bonding agent and 3 M Z ‘100’, composite resin. Impact strength of fragment bonded teeth and intact teeth tested with impact testing machine and compared. Results: Mean impact strength of fragment bonded teeth (30.76 KJ/M2 ) is not statistically significant deferent from mean impact strength of intact teeth (31.11 KJ/M2 ). Conclusion: Mean impact strength of fragment bonded teeth is not statistically different with that of intact teeth. Hence, after fracture of teeth if it is restored with fragment reattachment by using 3 M single bond dentin bonding agent and 3 M Z ‘100’ composite resin is having impact strength like that of intact teeth. How to cite the article: Venugopal L, Lakshmi MN, Babu DA, Kiran VR. Comparative evaluation of impact strength of fragment bonded teeth and intact teeth: An in vitro study. J Int Oral Health 2014;6(3):73-6. PMID:25083037

Oligodeoxynucleotide Probes for Detecting Intact Cells

NASA Technical Reports Server (NTRS)

Rosson, Reinhardt A.; Maurina-Brunker, Julie; Langley, Kim; Pynnonen, Christine M.

2004-01-01

A rapid, sensitive test using chemiluminescent oligodeoxynucleotide probes has been developed for detecting, identifying, and enumerating intact cells. The test is intended especially for use in detecting and enumerating bacteria and yeasts in potable water. As in related tests that have been developed recently for similar purposes, the oligodeoxynucleotide probes used in this test are typically targeted at either singlecopy deoxyribonucleic acid (DNA) genes (such as virulence genes) or the multiple copies (10,000 to 50,000 copies per cell) of 16S ribosomal ribonucleic acids (rRNAs). Some of those tests involve radioisotope or fluorescent labeling of the probes for reporting hybridization of probes to target nucleic acids. Others of those tests involve labeling with enzymes plus the use of chemiluminescent or chromogenic substrates to report hybridization via color or the emission of light, respectively. The present test is of the last-mentioned type. The chemiluminescence in the present test can be detected easily with relatively simple instrumentation. In developing the present test, the hybridization approach was chosen because hybridization techniques are very specific. Hybridization detects stable, inheritable genetic targets within microorganisms. These targets are not dependent on products of gene expression that can vary with growth conditions or physiological states of organisms in test samples. Therefore, unique probes can be designed to detect and identify specific genera or species of bacteria or yeast (in terms of rRNA target sequences) or can be designed to detect and identify virulence genes (genomic target sequences). Because of the inherent specificity of this system, there are few problems of cross-reactivity. Hybridization tests are rapid, but hybridization tests now available commercially lack sensitivity; typically, between 10(exp 6) and 10(exp 7) cells of the target organism are needed to ensure a reliable test. Consequently, the numbers of

Penetration of Ufenamate into Intact, Stripped, or Delipidized Skin Using Different Vehicles.

PubMed

Iino, Hayato; Fujii, Makiko; Fujino, Manami; Koizumi, Naoya; Watanabe, Yoshiteru

2015-01-01

The purpose of this study was to clarify the effect of skin condition on skin penetration of the very high lipophilic drug, ufenamate (UF). UF was applied to stripped or delipidized skin using liquid paraffin (LP) or purified water containing polysorbate 80 at a dose of 2 µL/cm(2). We found that UF penetration into intact and stripped skin using a water vehicle was respectively 5 and 10 times higher than that using LP. UF is freely soluble in oil and insoluble in water; thus, activity in water is higher than that in LP. Therefore, it is useful to use a water-based vehicle for both intact sites and those with defective stratum corneum (SC). Conversely, we found that delipidization of SC decreased the penetration of UF significantly with both LP and water, and the amount measured in the epidermis was 1 µg/cm(2) with both vehicles. This indicates that UF is not suitable for so-called "dry skin." This study revealed clinically relevant differences in the penetration of UF into intact, stripped, or delipidized skin conditions.

Differences in School Behavior and Achievement between Children from Intact, Reconstituted, and Single-Parent Families.

ERIC Educational Resources Information Center

Featherstone, Darin R.; And Others

1992-01-01

Analyzed differences in school behavior and achievement among students (n=530) in grades six through nine from intact, reconstituted, and single-parent families. Students from intact, two-parent families had fewer absences and tardies, higher grade point averages, and fewer negative and more positive teacher behavioral ratings than did those from…

In situ imaging of the mouse cochlea using two-photon microscopy

NASA Astrophysics Data System (ADS)

Yang, Xin; Pu, Ye; Psaltis, Demetri; Stankovic, Konstantina M.

2013-04-01

Intracochlear imaging is of great interest clinically because cochlea is the central organ of hearing. However, intracochlear imaging is technologically challenging due to the cochlea's small size and encasement in bone. The state-of- the-art imaging techniques are not adequate for high resolution cellular imaging to establish diagnosis without destroying the cochlea. We report in situ imaging of intact mouse cochlea using endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. TPEF eliminates the need for exogenous labeling and eradicating the staining-induced artifacts. We used a natural, membranous opening into the cochlea, the round window, as the optical access to reach the organ of Corti, requiring no additional slicing or opening. Our approach provides the maximum non-invasiveness in the imaging process. TPEF exhibits strong contrast allowing deep imaging of mouse cochlea with cellular and even subcellular resolution. Inner hair cell, outer hair cell and supporting cell are clearly identifiable in TPEF images. Distinct morphological differences are observed between healthy and noise-exposed cochleae, allowing detection of specific, noise-induced pathologic changes. The TPEF images taken through the round window are correlated with the whole mount sections, verifying their reliability. Compared with one-photon excitation fluorescence (OPEF) confocal microscope and wide-field transmission microscope images taken under the same magnification and resolution, TPEF images demonstrate clear advantages in terms of sharpness, signal to noise ratio and contrast. These capabilities provide a working foundation for microendoscopy-based clinical diagnostics of sensorineural hearing loss.

Genetically Targeted All-Optical Electrophysiology with a Transgenic Cre-Dependent Optopatch Mouse

PubMed Central

Lou, Shan; Adam, Yoav; Weinstein, Eli N.; Williams, Erika; Williams, Katherine; Parot, Vicente; Kavokine, Nikita; Liberles, Stephen; Madisen, Linda; Zeng, Hongkui

2016-01-01

Recent advances in optogenetics have enabled simultaneous optical perturbation and optical readout of membrane potential in diverse cell types. Here, we develop and characterize a Cre-dependent transgenic Optopatch2 mouse line that we call Floxopatch. The animals expressed a blue-shifted channelrhodopsin, CheRiff, and a near infrared Archaerhodopsin-derived voltage indicator, QuasAr2, via targeted knock-in at the rosa26 locus. In Optopatch-expressing animals, we tested for overall health, genetically targeted expression, and function of the optogenetic components. In offspring of Floxopatch mice crossed with a variety of Cre driver lines, we observed spontaneous and optically evoked activity in vitro in acute brain slices and in vivo in somatosensory ganglia. Cell-type-specific expression allowed classification and characterization of neuronal subtypes based on their firing patterns. The Floxopatch mouse line is a useful tool for fast and sensitive characterization of neural activity in genetically specified cell types in intact tissue. SIGNIFICANCE STATEMENT Optical recordings of neural activity offer the promise of rapid and spatially resolved mapping of neural function. Calcium imaging has been widely applied in this mode, but is insensitive to the details of action potential waveforms and subthreshold events. Simultaneous optical perturbation and optical readout of single-cell electrical activity (“Optopatch”) has been demonstrated in cultured neurons and in organotypic brain slices, but not in acute brain slices or in vivo. Here, we describe a transgenic mouse in which expression of Optopatch constructs is controlled by the Cre-recombinase enzyme. This animal enables fast and robust optical measurements of single-cell electrical excitability in acute brain slices and in somatosensory ganglia in vivo, opening the door to rapid optical mapping of neuronal excitability. PMID:27798186

Sex-comparative study of mouse cerebellum physiology under adult-onset hypothyroidism: The significance of GC-MS metabolomic data normalization in meta-analysis.

PubMed

Maga-Nteve, Christoniki; Vasilopoulou, Catherine G; Constantinou, Caterina; Margarity, Marigoula; Klapa, Maria I

2017-01-15

A systematic data quality validation and normalization strategy is an important component of the omic profile meta-analysis, ensuring comparability of the profiles and exclusion of experimental biases from the derived biological conclusions. In this study, we present the normalization methodology applied on the sets of cerebellum gas chromatography-mass spectrometry metabolic profiles of 124days old male and female animals in an adult-onset-hypothyroidism (AOH) mouse model before combining them into a sex-comparative analysis. The employed AOH model concerns the monitoring of the brain physiology of Balb/cJ mice after eight-week administration of 1%w/v KClO 4 in the drinking water, initiated on the 60th day of their life. While originating from the same animal study, the tissues of the two sexes were processed and their profiles acquired and analyzed at different time periods. Hence, the previously published profile set of male mice was first re-annotated based on the presently available resources. Then, after being validated as acquired under the same analytical conditions, both profiles sets were corrected for derivatization biases and filtered for low-confidence measurements based on the same criteria. The final normalized 73-metabolite profiles contribute to the currently few available omic datasets of the AOH effect on brain molecular physiology, especially with respect to sex differentiation. Multivariate statistical analysis indicated one (unknown) and three (succinate, benzoate, myristate) metabolites with significantly higher and lower, respectively, cerebellum concentration in the hypothyroid compared to the euthyroid female mice. The respective numbers for the males were two and 24. Comparison of the euthyroid cerebellum metabolic profiles between the two sexes indicated 36 metabolites, including glucose, myo- and scyllo-inositol, with significantly lower concentration in the females versus the males. This implies that the female mouse cerebellum has

Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

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

Wu, Si; Brown, Joseph N.; Tolic, Nikola

There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intactmore » N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.« less

Meta-analysis of peak wall stress in ruptured, symptomatic and intact abdominal aortic aneurysms.

PubMed

Khosla, S; Morris, D R; Moxon, J V; Walker, P J; Gasser, T C; Golledge, J

2014-10-01

Abdominal aortic aneurysm (AAA) is an important cause of sudden death; however, there are currently incomplete means to predict the risk of AAA rupture. AAA peak wall stress (PWS) can be estimated using finite element analysis (FEA) methods from computed tomography (CT) scans. The question is whether AAA PWS can predict AAA rupture. The aim of this systematic review was to compare PWS in patients with ruptured and intact AAA. The MEDLINE database was searched on 25 May 2013. Case-control studies assessing PWS in asymptomatic intact, and acutely symptomatic or ruptured AAA from CT scans using FEA were included. Data were extracted independently. A random-effects model was used to calculate standard mean differences (SMDs) for PWS measurements. Nine studies assessing 348 individuals were identified and used in the meta-analysis. Results from 204 asymptomatic intact and 144 symptomatic or ruptured AAAs showed that PWS was significantly greater in the symptomatic/ ruptured AAAs compared with the asymptomatic intact AAAs (SMD 0·95, 95 per cent confidence interval 0·71 to 1·18; P < 0·001). The findings remained significant after adjustment for mean systolic blood pressure, standardized at 120 mmHg (SMD 0·68, 0·39 to 0·96; P < 0·001). Minimal heterogeneity between studies was noted (I(2)  = 0 per cent). This study suggests that PWS is greater in symptomatic or ruptured AAA than in asymptomatic intact AAA. © 2014 BJS Society Ltd. Published by John Wiley & Sons Ltd.

Apoptosis of mouse hippocampal cells induced by Taenia crassiceps metacestode factor.

PubMed

Zepeda, N; Solano, S; Copitin, N; Chávez, J L; Fernández, A M; García, F; Tato, P; Molinari, J L

2017-03-01

Seizures, headache, depression and neurological deficits are the signs and symptoms most frequently reported in human neurocysticercosis. However, the cause of the associated learning and memory deficits is unknown. Here, we used Taenia crassiceps infection in mice as a model of human cysticercosis. The effects of T. crassiceps metacestode infection or T. crassiceps metacestode factor (MF) treatment on mouse hippocampal cells were studied; control mice were included. At 45 days after infection or treatment of the mice with MF, all mice were anaesthetized and perfused transcardially with saline followed by phosphate-buffered 10% formalin. Then the brains were carefully removed. Coronal sections stained using several techniques were analysed. Extensive and significant apoptosis was found in the experimental animals, mainly in the dentate gyrus, CA1, CA2, CA3 and neighbouring regions, in comparison with the apparently intact cells from control mice (P < 0.01). These results suggest that neurological deficits, especially the learning and memory deficits, may be generated by extensive apoptosis of hippocampal cells.

Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs

PubMed Central

Barrera, Leah O.; Li, Zirong; Smith, Andrew D.; Arden, Karen C.; Cavenee, Webster K.; Zhang, Michael Q.; Green, Roland D.; Ren, Bing

2008-01-01

By integrating genome-wide maps of RNA polymerase II (Polr2a) binding with gene expression data and H3ac and H3K4me3 profiles, we characterized promoters with enriched activity in mouse embryonic stem cells (mES) as well as adult brain, heart, kidney, and liver. We identified ∼24,000 promoters across these samples, including 16,976 annotated mRNA 5′ ends and 5153 additional sites validating cap-analysis of gene expression (CAGE) 5′ end data. We showed that promoters with CpG islands are typically non-tissue specific, with the majority associated with Polr2a and the active chromatin modifications in nearly all the tissues examined. By contrast, the promoters without CpG islands are generally associated with Polr2a and the active chromatin marks in a tissue-dependent way. We defined 4396 tissue-specific promoters by adapting a quantitative index of tissue-specificity based on Polr2a occupancy. While there is a general correspondence between Polr2a occupancy and active chromatin modifications at the tissue-specific promoters, a subset of them appear to be persistently marked by active chromatin modifications in the absence of detectable Polr2a binding, highlighting the complexity of the functional relationship between chromatin modification and gene expression. Our results provide a resource for exploring promoter Polr2a binding and epigenetic states across pluripotent and differentiated cell types in mammals. PMID:18042645

Adult-Onset Fluoxetine Treatment Does Not Improve Behavioral Impairments and May Have Adverse Effects on the Ts65Dn Mouse Model of Down Syndrome

PubMed Central

Heinen, Markus; Hettich, Moritz M.; Ryan, Devon P.; Schnell, Susanne; Paesler, Katharina; Ehninger, Dan

2012-01-01

Down syndrome is caused by triplication of chromosome 21 and is associated with neurocognitive phenotypes ranging from severe intellectual disability to various patterns of more selective neuropsychological deficits, including memory impairments. In the Ts65Dn mouse model of Down syndrome, excessive GABAergic neurotransmission results in local over-inhibition of hippocampal circuits, which dampens hippocampal synaptic plasticity and contributes to cognitive impairments. Treatments with several GABAA receptor antagonists result in increased plasticity and improved memory deficits in Ts65Dn mice. These GABAA receptor antagonists are, however, not suitable for clinical applications. The selective serotonin reuptake inhibitor fluoxetine, in contrast, is a widely prescribed antidepressant that can also enhance plasticity in the adult rodent brain by lowering GABAergic inhibition. For these reasons, we wondered if an adult-onset 4-week oral fluoxetine treatment restores spatial learning and memory impairments in Ts65Dn mice. Fluoxetine did not measurably improve behavioral impairments of Ts65Dn mice. On the contrary, we observed seizures and mortality in fluoxetine-treated Ts65Dn mice, raising the possibility of a drug × genotype interaction with respect to these adverse treatment outcomes. Future studies should re-address this in larger animal cohorts and determine if fluoxetine treatment is associated with adverse treatment effects in individuals with Down syndrome. PMID:22848851

Intact suppression of increased false recognition in schizophrenia.

PubMed

Weiss, Anthony P; Dodson, Chad S; Goff, Donald C; Schacter, Daniel L; Heckers, Stephan

2002-09-01

Recognition memory is impaired in patients with schizophrenia, as they rely largely on item familiarity, rather than conscious recollection, to make mnemonic decisions. False recognition of novel items (foils) is increased in schizophrenia and may relate to this deficit in conscious recollection. By studying pictures of the target word during encoding, healthy adults can suppress false recognition. This study examined the effect of pictorial encoding on subsequent recognition of repeated foils in patients with schizophrenia. The study included 40 patients with schizophrenia and 32 healthy comparison subjects. After incidental encoding of 60 words or pictures, subjects were tested for recognition of target items intermixed with 60 new foils. These new foils were subsequently repeated following either a two- or 24-word delay. Subjects were instructed to label these repeated foils as new and not to mistake them for old target words. Schizophrenic patients showed greater overall false recognition of repeated foils. The rate of false recognition of repeated foils was lower after picture encoding than after word encoding. Despite higher levels of false recognition of repeated new items, patients and comparison subjects demonstrated a similar degree of false recognition suppression after picture, as compared to word, encoding. Patients with schizophrenia displayed greater false recognition of repeated foils than comparison subjects, suggesting both a decrement of item- (or source-) specific recollection and a consequent reliance on familiarity in schizophrenia. Despite these deficits, presenting pictorial information at encoding allowed schizophrenic subjects to suppress false recognition to a similar degree as the comparison group, implying the intact use of a high-level cognitive strategy in this population.

Multitasking in older adults with type 2 diabetes: A cross-sectional analysis

PubMed Central

McDowd, Joan M.; Mahnken, Jonathan D.; Burns, Jeffrey M.; Sabus, Carla H.; Britton-Carpenter, Amanda J.; Utech, Nora B.; Kluding, Patricia M.

2017-01-01

Background and purpose Deficits in the ability to multitask contribute to gait abnormalities and falls in many at-risk populations. However, it is unclear whether older adults with type 2 diabetes mellitus (DM) also demonstrate impairments in multitasking. The purpose of this study was to compare multitasking performance in cognitively intact older adults with and without DM and explore its relationship to measures of gait and functional ability. Methods We performed a cross-sectional analysis of 40 individuals aged 60 and older with type 2 DM and a matched group of 40 cognitively intact older adults without DM. Multitasking was examined via the ambulatory Walking and Remembering Test (WART) and seated Pursuit Rotor Test (PRT). Self-selected normal and fast walking speed and stride length variability were quantitatively measured, and self-reported functional ability was assessed via the Late Life Function and Disability Index (LLFDI). Results Participants with DM walked slower and took more steps off path when multitasking during the WART. No between-group differences in multitasking performance were observed on the PRT. Multitasking performance demonstrated little correlation with gait and functional ability in either group. Discussion and conclusions Older adults with DM appear to perform poorly on an ambulatory measure of multitasking. However, we analyzed a relatively small, homogenous sample of older adults with and without type 2 DM and factors such as peripheral neuropathy and the use of multiple comparisons complicate interpretation of the data. Future research should explore the interactions between multitasking and safety, fall risk, and function in this vulnerable population. Clinicians should recognize that an array of factors may contribute to gait and physical dysfunction in older adults with type 2 diabetes, and be prepared to assess and intervene appropriately. PMID:29045492

EMMPRIN (basigin/CD147) expression is not correlated with MMP activity during adult mouse mammary gland development.

PubMed

Szymanowska, Malgorzata; Hendry, Kay A K; Robinson, Claire; Kolb, Andreas F

2009-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN/basigin/CD147) is a cell surface protein, which has been associated with the induction of matrix metalloproteinase (MMP) genes during cancer metastasis. EMMPRIN plays a role in a variety of physiological processes as is evident by the diverse deficiencies detectable in EMMPRIN knockout mice. We have analysed the role of EMMPRIN in the induction of MMP genes during mammary gland differentiation and involution. Co-transfection studies showed that EMMPRIN has diverse effects on MMP promoter activity in different mammary and non-mammary cell lines. Expression of EMMPRIN mRNA is enhanced markedly by insulin in a mammary gland cell line but appears to have no direct effect on MMP gene expression in these cells. Microarray analysis and quantitative PCR show that EMMPRIN is expressed throughout mammary gland differentiation in the mouse. Its expression decreases during early pregnancy and briefly after induction of mammary gland involution by litter removal. Immunohistochemical analysis shows that EMMPRIN expression is limited to the stromal compartment during pregnancy, whereas it is strongly expressed in the epithelium during lactation. In summary the data argue against a causal role for EMMPRIN for the induction of MMP gene expression during adult mammary gland development. These data therefore support a physiological role for EMMPRIN other than MMP induction in mammary gland biology. 2008 Wiley-Liss, Inc.

Demonstration of pelvic anatomy by modified midline transection that maintains intact internal pelvic organs.

PubMed

Steinke, Hanno; Saito, Toshiyuki; Herrmann, Gudrun; Miyaki, Takayoshi; Hammer, Niels; Sandrock, Mara; Itoh, Masahiro; Spanel-Borowski, Katharina

2010-01-01

Gross dissection for demonstrating anatomy of the human pelvis has traditionally involved one of two approaches, each with advantages and disadvantages. Classic hemisection in the median plane through the pelvic ring transects the visceral organs but maintains two symmetric pelvic halves. An alternative paramedial transection compromises one side of the bony pelvis but leaves the internal organs intact. The authors propose a modified technique that combines advantages of both classical dissections. This novel approach involves dividing the pubic symphysis and sacrum in the median plane after shifting all internal organs to one side. The hemipelvis without internal organs is immediately available for further dissection of the lower limb. The hemipelvis with intact internal organs is ideal for showing the complex spatial relationships of the pelvic organs and vessels relative to the intact pelvic floor.

10. VIEW SHOWING FRAGMENTED AND INTACT ORIGINAL SIDEWALK ALONG SOUTH ...

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

10. VIEW SHOWING FRAGMENTED AND INTACT ORIGINAL SIDEWALK ALONG SOUTH SIDE OF SHERMAN CANAL, BETWEEN DELL AVENUE VEHICULAR BRIDGE AND GRAND CANAL COURT PEDESTRIAN BRIDGE, LOOKING EAST - Venice Canals, Community of Venice, Los Angeles, Los Angeles County, CA

Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

PubMed Central

Hyung, Suk-Joon; Ruotolo, Brandon T.

2013-01-01

Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

[Effect of Tribulus terrestris extract on melanocyte-stimulating hormone expression in mouse hair follicles].