Search PubMed⌕ Search

Biomedical subjects

R Reynolds

Publications and source records attributed to R Reynolds.

At least 73 records · Page 4Linked to original sources
PubMed1996

An immunocytochemical study of glutamate receptors and glutamine synthetase in the hippocampus of rats injected with kainate.

Immunocytochemistry was used to study the distribution of the kainate receptors GluR1, GluR2/3 and GluR4 and of the N-methyl-D-aspartate (NMDA) receptor NMDAR1 as well as the astrocyte markers glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) in the hippocampus of normal and kainate-lesioned rats. Hippocampal pyramidal neurons and dentate granule neurons were labelled heavily for GluR1 and GluR2/3, but only lightly for GluR4. Dense GluR4 immunopositivity was, however, observed in oligodendrocyte-like glial cells. Hippocampal pyramidal neurons and dentate granule neurons were moderately labelled for NMDAR1. Intravenous kainate injections resulted in a decrease in GluR1 and GluR2/3 immunoreactivity on the apical dendrites of pyramidal neurons as early as 7 h postinjection. At 18 h, there was a marked reduction in GluR1 and GluR2/3 receptors in the terminal tuft of dendrites of most hippocampal pyramidal neurons in the affected area, although some cells showed labelling in other portions of the apical dendrites and in basal dendrites. Immunostaining for GluR4 and NMDAR1 was also reduced at this time. At postinjection day 3, only the cell bodies and the basal dendrites of a few scattered pyramidal cells were labelled. Taken together, these results indicate a progressive loss of glutamate receptors, which affects the apical dendritic tree before the basal dendritic tree. The decrease in receptor immunoreactivity could be due to a downregulation of the receptors, since it occurred as early as 7 h postlesion, before cell death was evident in Nissl-stained sections. At long intervals after kainate injection, all pyramidal cells at the centre of the lesion showed a lack of glutamate receptor staining, and no partially labelled pyramidal cells were observed. The periphery of the lesion, however, contained many partially labelled pyramidal neurons among the unlabelled cells and had features of early lesions. The present study also showed an early decrease in GS immunoreactivity in the affected CA fields of the hippocampus (18 h to 3 days postinjection), followed by a medium-term increase (5-68 days) and a late decrease in GS immunoreactivity (81 days). The decrease in GS immunoreactivity at 81 days is not due to an absence of astrocytes, since GFAP staining showed many densely labelled astrocytes in the affected CA field.

Animals↗
PubMed1996

Histochemistry of NADPH-diaphorase--a marker for neuronal nitric oxide synthase--in the carp retina.

The nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemical technique was used as a marker to assess the distribution of nitric oxide synthase activity in the carp retina. NADPH-diaphorase activity was found to be present in photoreceptors (rods and cones), horizontal cells, amacrine cells, bipolar cells, Müller cells and ganglion cells. Staining was most prominent in the photoreceptor ellipsoids but was not confined to any particular subtype. The density of the staining within the inner plexiform layer (IPL) was determined by image analysis. There was a broad peak of activity in each sublamina of the IPL, but sublamina b appeared to be relatively more heavily stained. The results taken together suggest that the nitric oxide signalling system could have a broader involvement in retinal function than previously thought. Furthermore, nitric oxide may have a novel mode of action in the retina whereby it could be effective on cells (photoreceptors) that also synthesize it.

Animals↗
PubMed1996

GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors.

We show that GGF/neuregulin is a mitogen for prooligodendrocytes (O4+/O1- cells), oligodendrocytes (O4+/O1+ cells), and type-2 astrocytes. Heregulin beta 1, another neuregulin isoform, is also mitogenic. The proliferative effect of glial growth factor (GGF) does not require, but is greatly potentiated by, serum factors. GGF also promotes the survival of pro-oligodendrocytes under serum-free conditions. High levels of GGF reversibly inhibit the differentiation and lineage commitment of oligodendrocyte progenitors and, in differentiated cultures, result in loss of O1 and myelin basic protein expression. All three erbB receptors are expressed by progenitors and are activated by GGF; the relative abundance of these receptors changes during differentiation. Finally, cortical neurons release a soluble mitogen for pro-oligodendrocytes that is specifically blocked by antibodies to GGF. These results implicate the neuregulins in the neuronal regulation of oligodendrocyte progenitor proliferation, survival, and differentiation.

Animals↗
PubMed1996

Microvascular anastomoses. A comparative study of fibrinogen adhesive and interrupted suture techniques.

A modified sleeve technique was developed for making microsurgical anastomoses using a commercially produced fibrinogen adhesive called Tisseel. A controlled study was then carried out to compare the new fibrinogen adhesive anastomoses with conventional suture anastomoses in a bilateral groin flap model using 50 consecutive rabbits. Statistical analysis of the results indicated that flap survival rate and vascular patency rate were comparable for the two techniques. The fibrinogen adhesive anastomoses took less time to complete and, subjectively, were less difficult technically. The suture anastomoses were more versatile. Histologic studies revealed that the adhesive did not flow through the sleeve into the lumen, and that, although there was a brief inflammatory response associated with healing, this inflammation was very localized and did not involve the inner layers of the vessel wall or lumen. It was concluded that the new technique was a useful addition to techniques already available.

Anastomosis, Surgical↗
PubMed1996

Post-amplification primer extension of heat-denatured AmpliType PCR products: effects on typing results.

Alleles of the HLA, DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobilized on a nylon membrane strip. Using reagents supplied in AmpliType PCR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequently, the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensities. The relative probe signal intensities obtained from an undegraded DNA sample extracted from a single individual will be different from those obtained from degraded DNA and from samples containing DNA from more than one source. Because probe signal intensity is an essential consideration for interpretation, factors that can influence it need to be identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signal intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the selective loss of GC B and HLA DQA1 4.1 probe signals observed after shorter cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficient residual Taq DNA polymerase activity to extend primers as the solution cools and that this primer extension occurs at a more rapid rate than PCR product reannealing. Primer extension across probe binding sites will prevent hybridization of the PCR product to complementary probes on the strip. The extent of signal reduction is dependent on the position of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all probe binding sites will be available for hybridization to PM and HLA DQA1 DNA probe strips.

DNA↗
PubMed1996

Gender determination of forensic samples using PCR amplification of ZFX/ZFY gene sequences.

Determining the gender of an evidentiary sample can be an important part of casework analyses. Gender information, particularly when combined with mitochondrial DNA analysis, can serve to distinguish biological evidence from two people who share the same DNA type(s) but differ by sex. When typing sexual assault evidence, gender information can serve as confirmation that the "sperm fraction" extracted from swabs and stains actually contains male DNA and also as an indicator of the amount of male DNA present in the non-sperm fraction. The PCR-based assay described here relies on amplification of a small, polymorphic region of a homologous zinc finger protein locus present on the X and Y chromosomes. The gender of the sample donor is determined from the PCR product either by Haelll restriction enzyme digestion followed by gel electrophoresis or by hybridization to immobilized sequence specific oligonucleotide probes (reverse dot blot). When using the reverse dot blot approach, amplification and typing of the gender PCR product can be coupled to amplification and typing of the AmpliType HLA DQalpha and PM markers. Sensitivity and mixture studies were performed in addition to the analysis of casework bloodstains and sexual assault kit samples. Additional studies using this gender determination assay are described in the accompanying paper.

Base Sequence↗
PubMed1995

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy.

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.

Animals↗
PubMed1995

Localization of glial fibrillary acidic protein and glutamine synthetase in the human cerebral cortex and subcortical white matter--a double immunolabelling and electron microscopic study.

Astrocytes in the human cerebral cortex and subcortical white matter were labelled using antibodies to glial fibrillary acidic protein, or double labelled for glial fibrillary acidic protein and glutamine synthetase, and studied by light and electron microscopy. We described two types of astrocytic processes in an earlier study of human cortex: glial-filament-rich but mitochondria-poor, and filament-poor but mitochondria-rich. The question arose as to whether these were different segments of the same process, or whether the filament-rich processes belonged to a subpopulation of 'fibrous' astrocytes. In the present study no such fibrous astrocytes were found in histologically normal cortex, and all glial fibrillary acidic protein positive cell bodies in the cortex were also glutamine synthetase-positive, and had features of protoplasmic astrocytes. Since the processes of fibrous astrocytes in the white matter seldom extended more than 40-50 microns beyond the cell bodies, they were unlikely to account for the filament-rich astrocytic processes that were observed in the cortex by electron microscopy. The filament-rich but mitochondria-poor, and the filament-poor but mitochondria-rich processes are seen in continuity in the cortex and must therefore both stem from cell bodies with features of protoplasmic astrocytes.

Adolescent↗
PubMed1995

AmpliType PM and HLA DQ alpha typing from pap smear, semen smear, and postcoital slides.

Deoxyribonucleic acid (DNA) samples extracted from stained pap smear, semen smear and postcoital slides were amplified by the polymerase chain reaction (PCR) and typed for the AmpliType PM and HLA DQ alpha alleles. HLA DQ alpha and PM types consistent with blood controls from the donors were obtained. Stained cells fixed to slides can provide a valuable source of material for determining a genetic profile of the sample donor, particularly in sexual assault cases and possibly in missing person cases involving women.

Coitus↗
PubMed1995

Consistency and reproducibility of AmpliType PM results between seven laboratories: field trial results.

The AmpliType PM Field Trial was designed to assess the ability of forensic laboratories to obtain the correct results from samples commonly encountered in forensic casework. The seven forensic laboratory participants of the AmpliType PM Field Trial each performed four studies. Samples were analyzed using components of the AmpliType PM PCR Amplification and Typing Kit. Laboratories were also provided with DNA probe strips to type the DQA1 locus. Of the 381 PM and 325 DQA1 DNA probe strip results obtained from DNA-containing and non-DNA-containing samples, 98.2% and 95.7% showed the correct result for PM and DQA1 types, respectively. No samples were typed incorrectly. The remaining small percentage of samples were either uninterpretable due to the presence of a mixture, or no result was obtained due to insufficient DNA. The Field Trial demonstrated that laboratories can easily implement the AmpliType PM system to analyze DNA-containing samples and controls successfully for forensic casework applications.

Body Fluids↗
PubMed1995

Validation of the AmpliFLP D1S80 PCR Amplification Kit for forensic casework analysis according to TWGDAM guidelines.

The validation of the AmpliFLP D1S80 PCR Amplification Kit for use in forensic casework was accomplished by performing all the relevant experiments outlined in the TWGDAM guidelines. Standard specimen and reproducibility studies were performed using organic and rapid DNA extraction techniques on both stain and liquid samples (blood, semen and saliva). Over 300 samples from three different populations (US Caucasians, African Americans and US Hispanics) were analyzed to determine allele and genotype frequencies. Purified DNA was mixed in defined ratios (ranging from unmixed DNA samples to 1:9 mixtures of 2 different DNA samples) prior to amplification to demonstrate that samples containing DNA from more than one individual can be detected and, in many cases, that the genotypes contributing to the mixture can be identified. Since casework samples frequently are exposed to environmental insults that can result in DNA degradation, purified DNA was degraded in the laboratory to analyze the effect of DNA fragment length on D1S80 amplification. It is crucial in the validation process to examine actual casework evidentiary material. This D1S80 kit can be used successfully by forensic scientists to amplify and type nonprobative evidentiary material, including bloodstains collected from crime scenes and rape kit materials collected for sexual assault cases. The D1S80 kit is specific to human DNA, and the D1S80 alleles are inherited according to the laws of Mendel. The sensitivity of the novel gel electrophoresis gel matrix allowed the PCR cycle number to be reduced to 29 cycles and the D1S80 kit sensitivity to be increased to 2.5 ng from the previous D1S80 Reagent Set specifications of 30 cycles and 5 ng, respectively.

Alleles↗
PubMed1994

The distribution of inflammatory demyelinated lesions in the central nervous system of rats with antibody-augmented demyelinating experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) has long been studied as an animal model of the human demyelinating disease Multiple Sclerosis. However, EAE induced in the Lewis rat by injection of myelin basic protein (MBP), or MBP-specific T-lymphocytes, is primarily an inflammatory condition of the central nervous system (CNS) with little or no demyelination. In EAE models in which demyelination does result, it is either not very widespread or is unpredictable in its degree and location. In this study we have produced antibody-augmented demyelinating EAE (ADEAE) in the Lewis rat by injection of activated MBP-specific T-lymphoblasts, followed by injection 4 days later of a monoclonal antibody against myelin/oligodendrocyte glycoprotein, an extrinsic protein of myelin. We have documented the extent and location of inflammatory cell infiltrates and demyelination throughout the CNS using histochemistry, immunofluorescence, and image analysis. Perivascular inflammatory infiltrates were seen in the deep cerebellar white matter and in the folia. Perivascular, periventricular, and subpial inflammation was widespread throughout the pons/medulla and at all levels of the spinal cord. Very little inflammation was apparent in the forebrain. MBP immunofluorescence demonstrated extensive areas of periventricular demyelination in the forebrain around the third ventricle. Both periventricular and perivascular lesions were commonly observed in the cerebellum and pons/medulla. The extent of demyelination in the spinal cord increased caudally with large confluent areas of subpial demyelination seen throughout the lumbar cord. The extensive and reproducible distribution of inflammatory demyelinating lesions in ADEAE provide the possibility to select areas of the CNS for more detailed analysis of the cellular changes that accompany demyelination and remyelination.

Animals↗
PubMed1994

Transdermal fentanyl disposition in elderly subjects.

The pharmacokinetic characteristics of a 20-cm2 fentanyl 24-hour transdermal patch were compared between 10 healthy elderly subjects 67-87 years and 6 young subjects 19-27 years. All 10 elderly subjects required patch removal prior to 24 h due to adverse effects versus none of the young subjects. The mean patch duration (PD) in elderly subjects was 11.7 +/- 4.9 h, yielding a mean area under the curve from 0 to 60 h (AUC0-60) of 20.4 +/- 10.3 ng h/ml versus a mean AUC0-60 of 21.0 +/- 10.4 ng h/ml in young subjects. Correcting AUC0-60 for PD (AUC0-60/PD) gave a mean value of 2.05 +/- 1.10 ng/ml for elderly subjects, which was significantly greater than the AUC0-60/PD of 0.88 +/- 0.44 ng/ml in young subjects (p = 0.034, Student's t test). The higher serum concentrations reflect increased absorption and/or decreased clearance in the elderly.

Administration, Cutaneous↗
PubMed1993

Cellular reaction to an acute demyelinating/remyelinating lesion of the rat brain stem: localisation of GD3 ganglioside immunoreactivity.

We describe a simple and reproducible acute demyelinating lesion of the rat brain stem induced by injection of ethidium bromide into the cisterna magna of young adult rats. Using immunofluorescence with a panel of antibodies to cell-specific antigens we have studied the changes in cell populations that occur at various stages during lesion progression and repair. In particular we localized the expression of ganglioside GD3 immunoreactivity, a marker for oligodendroglial progenitors in developing brain. Both astroglia (GFAP+) and oligodendroglia (CNP+) were destroyed during the early response to the ethidium bromide although axons were spared. Splitting of myelin lamellae occurred as early as 4 days post-injection (DPI), with extensive demyelination of the inferior cerebellar peduncle following by 6 DPI. Large numbers of ED1+ and OX-42+ macrophages were present in the lesion site at this stage. Astrogliosis occurred around the perimeter of the lesions. Two populations of GD3+ cells appeared within and around the lesion sites during the demyelination. One population was identified by the phenotype GD3+ ED1+ and thus probably belonged to the macrophage/microglial lineage. In these cells both antigens appeared cytoplasmic. The second population of GD3+ cells exhibited cell membrane GD3 immunoreactivity but did not express the ED1 antigen. These cells are suggested to be oligodendroglial progenitors generated in response to the demyelination. No such cells were seen in control tissue. GD3+ cells were present within the lesion sites from 6 DPI until 10-12 DPI. Following the clearance of myelin debris from the lesions, remyelination was a relatively rapid event with thin MBP+ myelin sheaths first seen at 11-12 DPI. Remyelination, which was extensive by 25 DPI, was predominantly oligodendroglial in origin (MBP+P0- myelin) with only small pockets of peripheral myelin (MBP+P0+ myelin) observed. The present study, in addition to identifying putative glial progenitors within a demyelinated lesion, also demonstrates the difficulties in unambiguously identifying such cells in the normal and damaged adult CNS.

Animals↗
PubMed1993

Neuron-oligodendroglial interactions during central nervous system development.

It is well established that a variety of growth factors influence the differentiation of oligodendroglial lineage cells in culture, although little information is available concerning the role and source of these factors in vivo. Developing oligodendroglia are almost constantly in a neuronal environment and would be expected to respond to a variety of signals from neurons that affect their survival, migration, division, maturation and myelin production. However, very little is known about the specific interactions that occur between these two cell types. Here we review the experimental evidence for the influence of neurons on oligodendroglial differentiation, including studies on the effects of both soluble factors and contact dependent events. We also propose a scheme for the control of myelinogenesis via both internal and external signals.

Animals↗
PubMed1993

Rat cerebral cortical neurons in primary culture release a mitogen specific for early (GD3+/04-) oligodendroglial progenitors.

The development of cells of the oligodendroglial lineage, from immature progenitor to myelinating cells, occurs largely in a neuronal environment, yet little is known about specific interactions between these 2 cell types. We have tested the effects of medium conditioned by cultures of rat cerebral cortical neurons (CCM), cerebellar granule interneurons (GCM), and a dorsal root ganglion derived cell line (NDCM) on cells of the oligodendroglial lineage in culture. Different stages of the lineage were defined using the cell surface antigens GD3, 04, and GalC. CCM and NDCM were mitogenic for the early GD3+/04- oligodendroglial progenitor, whereas GCM was mitogenic for the later GD3+/04+ stage. Neutralising antibodies to PDGF and bFGF were able to eliminate the mitogenic activity of NDCM and GCM, respectively, but did not abolish the mitogenic effect of CCM. We have demonstrated that neurons in primary culture from distinct CNS regions exert different influences on cells of the oligodendroglial lineage, and specifically that cortical neurons release an unknown mitogen for GD3+/04- oligodendroglial progenitors.

Animals↗
PubMed1993

Retarded development of neurons and oligodendroglia in rat forebrain produced by hyperphenylalaninemia results in permanent deficits in myelin despite long recovery periods.

The severe hypomyelination seen in the CNS of untreated phenylketonuria (PKU) patients has been suggested to be the result of a defect in the process of myelination itself. Using chronic hyperphenylalaninemia (HPA) in rats as a model of PKU we have previously shown, by immunohistochemistry, that axonal maturation as well as myelination was severely retarded. In the present study we have used image analysis techniques to quantitate changes in myelin basic protein (MBP) and 200-kDa neurofilament protein (NF-H) immunostaining in the corpus callosum and cerebral cortical grey matter of HPA rats. No difference in the density of MBP+ myelin was observed in the corpus callosum after 24 days HPA treatment although the width of the tract was much reduced. In contrast there was a deficit in NF-H immunostaining. Large deficits in both myelin and axonal maturity were seen in the cortical grey matter. Following a 6-week recovery period, despite recovery in the corpus callosum, large deficits in both MBP and NF-H were still seen in all cortical layers. Deficits in NF-H immunostaining were two to three times greater than those for MBP. On increasing the recovery period to 18 weeks significant deficits in myelin remained in layers I-III of the cortical grey matter whereas NF-H immunostaining had returned to normal levels in all layers. Our data suggest a primary effect of HPA on neuronal development, in particular axonal maturation, with a secondary hypomyelination and show that permanent deficits in myelinated axons in outer cortical layers can result when myelination is severely inhibited during a critical developmental period.

Animals↗