Biomedical subjects

Paul G Layer

Publications and source records attributed to Paul G Layer.

18 recordsLinked to original sources

PubMed2006

Naturally occurring mutation Leu307Pro of human butyrylcholinesterase in the Vysya community of India.

BACKGROUND: People with genetic variants of butyrylcholinesterase (EC 3.1.1.8, BChE) can have hours of prolonged apnea after a normal dose of succinylcholine or mivacurium. METHODS: Plasma samples from 226 people in the Vysya community in Coimbatore, India were tested for BChE activity. RESULTS: Nine unrelated individuals had no detectable activity. DNA sequencing revealed a novel mutation in exon 2 of the BCHE gene, responsible for the silent phenotype of human serum BChE. All silent BChE samples were homozygous for a point mutation at codon 307 (CTT-->CCT), resulting in substitution of leucine 307 by proline. Western blot analysis with a monoclonal antibody showed no BChE protein in plasma. Silent BChE plasma samples had no organophosphate-reactive BChE, as measured with FP-biotin. Expression of recombinant Leu307Pro BChE in cell culture confirmed that this mutant is expressed at very low levels. The proline substitution most likely destabilizes the BChE structure and causes the protein to be misfolded and rapidly degraded. CONCLUSIONS: This is the first report of a molecularly defined BChE mutation in the Indian population. The frequency of homozygous silent BChE in the Vysya community is 1 in 24, a value 4000-fold higher than the frequency of homozygous silent BChE in European and American populations.

Adolescent↗

PubMed2006

Knock-down of GFRalpha4 expression by RNA interference affects the development of retinal cell types in three-dimensional histiotypic retinal spheres.

PURPOSE: To determine the role of glial cell line-derived neurotropic factor family receptor alpha 4 (GFRalpha4) during retinogenesis in a three-dimensional histiotypic in vitro model of the embryonic chicken retina. METHODS: Retinal spheres were cultured from dissociated 6-day-old chicken retina under permanent rotation and transfected with GFRalpha4 siRNA at culture day 2. Alterations on proliferation, apoptosis, and differentiation were determined by semiquantitative RT-PCR, in situ hybridization, and immunohistochemistry after 24, 48, and 72 hours. RESULTS: In contrast to control cultures, retinal spheres transfected with GFRalpha4 siRNA showed reduced GFRalpha4 mRNA expression of only 38% after 24 hours, 3% after 48 hours, and 5% after 72 hours. Based on the suppression of GFRalpha4, a decline in proliferating cells from 10% to 4.8% even after 24 hours and a reduction of sphere size by up to 25% at later culture stages were observed. Moreover, the number of Pax 6-positive amacrine, ganglion, and horizontal cells was significantly decreased from 36% to 16% in GFRalpha4 siRNA-transfected retinal spheres 72 hours after transfection. Additionally, GFRalpha4 gene silencing affected the development of different types of photoreceptors, as revealed by a significant decrease of blue opsin mRNA expression from 29% to 2%, whereas green opsin mRNA and the number rho4D2-positive photoreceptors were significantly increased. CONCLUSIONS: These data showed for the first time that GFRalpha4 plays an essential role in regulating, at least in vitro, the development and differentiation of various cell types during retinogenesis.

Amacrine Cells↗

PubMed2006

On functions of cholinesterases during embryonic development.

Expression of cholinesterase (ChE) activity during phases of embryonic development is a general phenomenon in embryonic tissues. To elucidate the role(s) of ChEs during embryonic development, one line of research followed the assumption of a primitive muscarinic system involved in morphogenesis (Hohmann et al., 1995). This means that ChE functioning during development fits into the classical cholinergic neurotransmitter system: acetylcholine (ACh), as a signal, binds to ACh receptors and then is degraded by acetylcholinesterase (AChE) as the terminating enzyme. However, this is just one of the possible mechanisms. The other line of research was driven by evidence for noncholinergic functions of ChE proteins (AChE and butyrylcholinesterase [BChE]). There is accumulating data that other sites on AChE could exert nonclassical roles related to cell differentiation, neurite outgrowth, and adhesion.

Acetylcholinesterase↗

PubMed2005

Expression of acetylcholinesterase (AChE) and aryl acylamidase (AAA) during early zebrafish embryogenesis.

In zebrafish, the onset of acetylcholinesterase (AChE) expression was detected by RT-PCR at 4 hpf (hours post-fertilization). The aryl acylamidase (AAA) associated with AChE, a serotonin sensitive activity with unknown physiological function, was significantly higher than the esterase activity on zebrafish embryos homogenates at 4-12 h development (test-t = 3.523; d.f. = 4). Remarkably, the ratio of AAA/AChE activity decreased 210-fold from 4 to 144 h development, indicating a distinct embryonic role of AAA during early embryogenesis. The AAA activity was sensitive to eserine and serotonin, ensuring its association with AChE. This is the first report of AAA activity on fish, establishing zebrafish as a model to study AAA on development.

Acetylcholinesterase↗

PubMed2005

Mouse AChE binds in vivo to domain IV of laminin-1beta.

Functions of acetylcholinesterase (AChE) other than hydrolysis of acetylcholine, e.g. related to cell differentiation, synaptogenesis, neuronal signaling and diseases are well documented, but mechanisms supporting them are not understood. Therefore, we searched for AChE ligands in the central nervous system using a yeast two-hybrid screen (Y2H). 18 independent candidates were identified. One of the membrane or extracellular proteins was laminin-1, an extracellular matrix protein involved in neuronal differentiation and adhesion. The laminin-1 fragment found to interact with AChE contains 898 bp from the 1beta-chain. Moreover, we identified the region containing amino acid residues 240-503 of AChE as essential for interaction with laminin-1beta. Co-immunoprecipitation experiments confirmed the yeast two-hybrid results.

Acetylcholinesterase↗

PubMed2005

Lamina formation in the Mongolian gerbil retina (Meriones unguiculatus).

Retinae of nocturnal rodents, such as mice and rats, are almost exclusively rod-dominated. The gerbil, in contrast, shows active periods during day and night and uses both rod- and cone-based vision. However, its retina has not been studied in detail, except for one developmental study analysing its prenatal period (Wikler et al. 1989). Here, the formation of the laminar structure of the gerbil retina was studied from birth until late adult stages. At birth, the retina consisted of a wide neuroblastic layer, with 30% of cells still dividing, a rate decreasing to nearly zero by P6. Shortly after birth, segregation of a ganglion cell layer began. All retinal layers reached their final size around P20, as determined from DAPI-stained cryosections. Muller glial cells developed their typical structure from P1 onwards, e.g. announcing an outer plexiform layer (OPL) at P5, as analysed by the Ret-G7 and glutamine synthetase antibodies. The analyses of the inner retina were performed by antibodies to calretinin (CR) and calbindin (CB). CR is expressed in ganglion cells followed by amacrine cells from P1 onwards; their processes formed four subbands in the inner plexiform layer (IPL) and appeared sequentially after P5 until P20. CB stained a subtype of horizontal cells with their processes into the OPL from P14 onwards. The rod-specific antibody rho4D2 announced photoreceptors at P4, showing signs of outer segments from P10 onwards. The study shows that the formation of all retinal layers in the gerbil occurs postnatally. This and the fact that the gerbil retina is not exclusively rod-dominated could render the gerbil a valuable model for in vitro studies of retinogenesis in rodents.

Aging↗

PubMed2005

Rhodopsin, violet and blue opsin expressions in the chick are highly dependent on tissue and serum conditions.

The molecular, cellular or tissue environment can influence the expression of genes and thereby regulate processes of tissue formation. Here we determined the tissue and serum dependence of the expression of all photopigments in the chick by a series of distinct retinal cell cultures, analyzed by RT-PCR using specific primers for all four opsins and rhodopsin followed by quantitative scanning of the respective gel bands. For comparison, we first determined expression of all opsins during normal chick retinogenesis, which began with red and violet opsins at E12, shortly followed by blue and green opsins and finally rhodopsin at E14. This period corresponds to the time of synaptogenesis in the inner retina. All cultures were started with 6-day-old dissociated retinal cells. Cells were kept at low or high cell density (called LoDens or HiDens), or they were reaggregated as retinal spheres, whereby all of them were raised at low (2%) or high serum (12%) levels (called LoSer or HiSer). In LoDens/HiSer cultures, expression of all opsins was weak. At HiDens/LoSer red and green opsin expression was strong, while rhodopsin and violet/blue remained low. In HiDens/HiSer cultures the expression of red and green was strong; rhodopsin was almost normal, while violet and green were low. In reaggregates at high serum the expression came closest to a normal retina, but violet and blue opsins were still below normal. At low serum, however, violet and blue were negligible and rhodopsin was low. This in vitro study shows that rhodopsin, followed by violet and blue opsin expressions is highly dependent on serum, cell density and tissue conditions, while red and green opsins are more autonomous.

Animals↗

PubMed2004

Mouse acetylcholinesterase interacts in yeast with the extracellular matrix component laminin-1beta.

Acetylcholinesterase (AChE) is likely to have roles other than the hydrolysis of acetylcholine, e.g., related to developmental processes like neurite outgrowth, differentiation and adhesion. Here, we investigated whether AChE can function as a heterophilic cell adhesion molecule and searched for proteins interacting with it. Using the yeast two-hybrid method and a mouse brain cDNA library, we have identified an interaction between a partial cDNA encoding the globular domain IV of laminin chain beta1 and the amino acids 240-503 of mouse AChE. Biochemical co-immunoprecipitation assays confirmed the genetic results. We suggest that AChE, by interacting with laminin-1, is able to exert changes in adhesion signaling pathways.

Acetylcholinesterase↗

PubMed2004

Spatial and temporal expression patterns of GDNF family receptor alpha4 in the developing chicken retina.

GDNF family receptor alpha (GFRalpha) receptors are involved in the regulation of different aspects of embryonic development such as proliferation, migration, differentiation and survival. To determine the possible role of GFRalpha4 in retinal development, we analysed its expression in the developing chicken retina. We found that GFRalpha4 is temporally co-expressed with c-ret. Both, the temporal and spatial expression of GFRalpha4 is developmentally regulated during retinogenesis and is first detected in cells of the ganglion cell layer at E6. As development of the retina proceeds, the expression of GFRalpha4 extends to cells of the inner half of the inner nuclear layer and to cells of the outermost cell row of the inner nuclear layer. Later on, GFRalpha4 expression is also found in additional cells of the outer half of the inner nuclear layer and in a subpopulation of photoreceptors. A central-to-peripheral gradient of retinal differentiation is evident, as the onset of GFRalpha4 expression is first detectable in the central retina, while it is delayed by two days in its periphery.

Animals↗

PubMed2004

Aryl acylamidase activity on acetylcholinesterase is high during early chicken brain development.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to exhibit aryl acylamidase activities (here called AAA(AChe) and AAA(BChe), respectively), which have been suggested to be involved in developmental and pathological processes. We here have investigated the developmental profiles of both AAA(AChe) and AAA(BChe) activities along with their AChE and BChE activities from embryonic days E3 to hatching (E21) in Triton-extracted homogenates from chicken embryonic brains. AAA(AChe) follows continuously an increase that is typical for AChE expression itself, whereas AAA(BChe) was relatively high before E10 to then become negligible toward hatching. Sucrose gradient centrifugation of both homogenized and immunopurified samples from E6-E18 brains showed that all globular forms (G1, G2, G4) of AChE present AAA(AChe) activity. Interestingly, the ratio of AAA(AChe) to AChE is highest at E6, and here again higher on G1/G2- over the G4-form. Noticeably, the sensitivity of AAA(AChe) toward the specific AChE inhibitor BW284c51 at all stages is higher than that of AChE itself. These data of high ratios of AAA associated at young stages with cholinesterases strongly indicate a role of AAA in early brain development.

Acetylcholinesterase↗

PubMed2004

Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors.

Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration.

Acetylcholinesterase↗

PubMed2004

Expression of CNTF receptor-alpha in chick violet-sensitive cones with unique morphologic properties.

PURPOSE: Application of ciliary neurotrophic factor (CNTF) can rescue mature photoreceptors from lesion-induced and hereditary degeneration. In the chick retina, expression of the CNTF receptor is present in a subpopulation of photoreceptor cells. The present study was undertaken to identify the CNTF receptor-expressing photoreceptors and to describe the subcellular localization of the receptor protein. METHODS: The localization of the CNTF receptor was analyzed by light and electron microscopic immunocytochemistry in chick retinal wholemount preparations, with an antibody for CNTF receptor alpha (CNTFRalpha). Immunoreactive cells were identified by double labeling with immunocytochemical markers for photoreceptor subpopulations. RESULTS: The CNTFRalpha antibody labeled evenly distributed outer segments (OS) of a photoreceptor subpopulation. CNTFRalpha-positive OS were associated with oil droplets of uniform size. Receptor immunoreactivity did not colocalize with markers for rods and red-green cones. Complete overlap was found after double labeling with the antibody CERN 933, which recognizes violet-sensitive cones in the chick retina. Ultrastructurally, the CNTFRalpha-immunoreactive OS showed rodlike properties: an elongated shape and stacks of membrane discs separated from the plasma membrane. Immunoreactivity was completely restricted to the plasma membrane of the OS and the inner membrane sheet of the photoreceptor calices present in avian retinas. CONCLUSIONS: CNTFRalpha expression identifies a unique type of photoreceptors in the avian retina which does not fit into the classic morphologic definition of rods and cones. The specific expression in violet-sensitive photoreceptors suggests that CNTF may have a neuroprotective role related to the specific function of these cells.

Animals↗

PubMed2003

Transient muscarinic calcium mobilisation in transdifferentiating as in reaggregating embryonic chick retinae.

Two independent in vitro regeneration systems of the embryonic chick retina (E4-5) were used to study the mobilisation of intracellular calcium by the neurotransmitters acetylcholine (ACh) and glutamate, as measured by Fura-2 fluorescence changes. Retinal pigment epithelium (RPE) explants under the influence of basic fibroblast growth factor transdifferentiate into a retina-like tissue with normal laminar organisation, while rosetted spheres reaggregated from fully dispersed cells of the embryonic retina will achieve only an inferior tissue organisation, characterised by regions of an inverted retina [Layer et al., Neuroreport 12 (2001) A39-46]. ACh induced a pronounced Ca(2+) response in young explants, and a similar but less pronounced response in reaggregates; this response decreased almost entirely after 1 week in culture. In contrast, a Ca(2+) response to glutamate became detectable later, continuously increasing during this period. The response to ACh was strictly mediated by muscarinic ACh receptors (mAChRs), since it was inhibited by preincubation with atropine, but not tubocurarine; correspondingly, it was mimicked by muscarine, but not nicotine. Studies with mAChR blockers, preferentially acting on the m1-, m2-, or m3-receptor subtypes, suggested that the muscarine-induced Ca(2+) response is mediated by m1- and/or m3-type mAChRs, but not by the m2-type. These results show that (i) similar to the in vivo retina, in both a transdifferentiating and a reaggregating system an early muscarinic Ca(2+) response is active, which (ii) roughly parallels periods of cell proliferation, and (iii)-as evidenced by the reaggregates-does not depend on any tissue pre-organisation. In contrast, a response to glutamate becomes prominent only when tissue differentiation commences. This is the first demonstration of a physiological response in regenerating chick retinas, supporting their validity as models both of retinal development and regeneration.

Acetylcholine↗

PubMed2003

GDNF regulates chicken rod photoreceptor development and survival in reaggregated histotypic retinal spheres.

PURPOSE: To investigate the role of glial-cell-line-derived neurotrophic factor (GDNF) on proliferation, differentiation, and apoptosis of different retinal cell types--in particular, photoreceptor cells. METHODS: Reaggregated histotypic spheres, derived from retinal cells of the E6 chicken embryo were used. Under rotation, so-called rosetted spheroids arose by aggregation of dissociated retinal cells, followed by the proliferation, migration, differentiation and programmed cell death of particular cell types. Rosetted spheroids were cultured under serum-reduced conditions, either in the absence or presence of 50 ng/mL GDNF. At appropriate stages, rosetted spheroids were analyzed by using conventional staining and immunolabeling with antibodies against different retinal cell types. RESULTS: At early stages of culture, the application of GDNF to rosetted spheroids significantly increased and sustained the rate of proliferation. In particular, a de novo production of rod photoreceptors was observed, whereas cone photoreceptors and amacrine, horizontal, ganglion, and Müller cells were not affected. In addition, in GDNF-treated cultures, rod photoreceptors differentiated earlier than in nontreated cultures. In older rosetted spheroids raised in absence of GDNF, rod but not cone photoreceptors underwent apoptosis. By supplementation with GDNF, the percentage of dying rod photoreceptors was dramatically reduced (31%-6% at 8 days in culture, 71%-3% at 10 days in culture). Both the mitogenic and survival promoting effect of GDNF were dose dependent. CONCLUSIONS: The results strongly suggest that GDNF, at least in vitro, affects rod photoreceptors. Depending on the developmental stage, GDNF regulates their proliferation, differentiation, and survival.

Animals↗

PubMed2002

Non-antisense cellular responses to oligonucleotides.

Oligonucleotides induce various cellular responses which are not due to the blockade of protein synthesis by an antisense mechanism. Oligonucleotides presenting double-stranded or G-quartet structures (ribo- or deoxyribonucleotides, phosphodiester or phosphorothioated) induce retraction of neurites and aggregation of chicken retinal cells within 10-20 h. This effect is reversible, non-toxic; it appears to require internalization and can be mimicked by treatment of the cells with an RGDS peptide. The oligonucleotides appear to trigger a cascade of intracellular events, affecting the adhesive properties of integrins. In addition, a subset of oligonucleotides induced platelet aggregation, probably through their interaction with membrane receptors. Recognition of these effects is important for the design and interpretation of antisense experiments.

Animals↗

PubMed2002

Of layers and spheres: the reaggregate approach in tissue engineering.

The reaggregate approach involves the regeneration of histotypical three-dimensional spheres from dispersed cells of a given tissue in suspension culture. Reaggregated spheres are used as tumour, genetic, toxicological, biohybrid and neurosphere models, and often replace animal experimentation. A particularly instructive example is the use of reaggregation to regenerate complete laminar tissue from avian embryonic retina. By revealing constraints of layered tissue formation, such retinal spheres could be instrumental for regenerative medicine, including stem cell-based tissue engineering.

Animals↗

PubMed2002

Light-dark and circadian melatonin rhythms are established de novo in re-aggregates of the embryonic chicken retina.

A central function of chicken photoreceptors is the rhythmic synthesis of the neurohormone melatonin; however, it is unclear how they can achieve--already before hatching (i.e. without light exposure)--a circadian pacemaker system. Here we studied melatonin synthesis and secretion in rosetted spheroids, which are de novo histotypic spheres, re-aggregated from dissociated retinal cells of chicken embryos. Spheroids were cultured during a 12-hour light/12-hour dark cycle. Shortly after the onset of photoreceptor differentiation in spheroids near day 5 in culture, melatonin secretion set in and, by day 8 in culture, showed distinct dark-light oscillations, with high values during the dark and lower ones during the light period. Concomitantly, expression of two key enzymes of melatonin synthesis, arylalkylamine-N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected by northern blot analysis from day 6 onwards, increasing until day 8. Only HIOMT, but not AA-NAT levels were 2- to 3-fold higher at the end of the light compared with the dark phase. Even under permanent darkness, a slight melatonin oscillation was revealed after 6 days. In conclusion, photoreceptors in de novo regenerated retinal spheroids become light-responsive, establish appropriate pathways for melatonin synthesis and secrete more melatonin in the dark. The underlying circadian pacemaker seems to depend on photoreceptor differentiation, not on previous light-dark entrainment.

Acetylserotonin O-Methyltransferase↗

PubMedDate not supplied

Artificial design of three-dimensional retina-like tissue from dissociated cells of the mammalian retina by rotation-mediated cell aggregation.

The goal of this study was to establish a reliable three-dimensional culture system for the mammalian retina that allows the analysis of retinal function and dysfunction. To produce three-dimensional retinal tissues in vitro, dissociated retinal cells of neonatal rats were maintained in culture dishes on a self-made orbital shaker. On the basis of well-defined rotation conditions, dissociated free-floating cells reaggregate in the center of the culture dish to form a multicellular cluster. Subsequently, cells begin to proliferate, whereby they form spherelike retinal tissues that grow to a size of 180-210 microm. Immunohistochemical characterization of mature retinal spheres revealed the presence of ganglion cells, amacrine cells, Müller cells, and rod photoreceptors, which are arranged in different retina-like layers. Although a small number of cells undergo programmed cell death, retinal spheres remain viable for at least 35 days in culture as revealed by fluorescein diacetate and TUNEL staining. Because most biological processes involved in tissue organization such as proliferation, differentiation, apoptosis, and survival are also observable in retinal spheres, the presented novel mammalian three-dimensional culture system is not only an outstanding model for basic research but may also be of great benefit for stem cell tissue engineering and the pharmaceutical industry.

Animals↗