Biomedical subjects

M McKinney

Publications and source records attributed to M McKinney.

At least 19 recordsLinked to original sources

PubMed2002

Evaluation of cognitive assessment in stroke rehabilitation.

OBJECTIVE: To assess the effect of cognitive assessment on the functional outcome of stroke patients and quality of life for both patients and their carers. DESIGN: A multicentre, single-blind, randomized controlled trial. SETTING AND PARTICIPANTS: Two hundred and twenty-eight stroke patients were recruited from hospital wards in three UK centres. INTERVENTIONS: Patients were screened for cognitive impairment and randomly allocated to either routine care (116 patients) or routine care plus a detailed cognitive assessment (112 patients). MAIN OUTCOME MEASURES: Outcome was assessed three and six months after recruitment by an independent assessor blind to the intervention on Extended ADL, Cognitive Failures Questionnaire, General Health Questionnaire-28 for patients and carers and Carer Strain Index. RESULTS: There was no significant difference between the two groups in patients' functional outcome, perceived cognitive ability, level of psychological distress or satisfaction with care. There was a trend for the assessment group to have lower levels of carer strain (p = 0.06). CONCLUSIONS: The provision of information about cognitive assessment in stroke rehabilitation may decrease carer strain.

Aged↗

PubMed2001

Survival and plasticity of basal forebrain cholinergic systems in mice transgenic for presenilin-1 and amyloid precursor protein mutant genes.

The basalo-cortical cholinergic system was characterized in mice expressing mutant human genes for presenilin-1 (PS1), amyloid precursor protein (APP), and combined PS/APP. Dual immunocytochemistry for ChAT and A beta revealed swollen cholinergic processes within cortical plaques in both APP and PS/APP brains by 12 months, suggesting aberrant sprouting or redistribution of cholinergic processes in response to amyloid deposition. At 8 months, cortical and subcortical ChAT activity was normal (PS/APP) or elevated (PS, APP frontal cortex), while cholinergic cell counts (nBM/SI) and receptor binding were unchanged. ChAT mRNA was up-regulated in the nBM/SI of all three transgenic lines at 8 months. The data indicate that the basal forebrain cholinergic system does not degenerate in mice expressing AD-related transgenes, even in mice with extreme amyloid load. The

Acetylcholine↗

PubMed2001

Visualization of muscarinic receptor-mediated phosphoinositide turnover in the hippocampus of young and aged, learning-impaired Long Evans rats.

Hippocampal receptor-mediated phosphoinositide (PI) turnover is severely blunted in aged rats that demonstrate cognitive deficits in the Morris water maze. To further examine the anatomical localization of this deficit, we examined the topography of muscarinic receptor-mediated PI turnover in young and aged-learning impaired rats by taking advantage of an autoradiographic method that visualizes PI turnover by measuring the diacylglycerol (DAG) branch of the PI turnover signal transduction system. Using this method, muscarinic cholinergic receptors were stimulated in hippocampal slices with agonist, and the receptor-mediated incorporation of [3H] cytidine into [3H]CDP-DAG was subsequently quantified in subregions of the hippocampus using film autoradiography. Our results show a significant decrease in basal incorporation of [ 3H]CDP-DAG in the subiculum and in the dentate gyrus in the aged rats. The muscarinic receptor-mediated [3H]CDP-DAG response was significantly blunted in the aged rats in subiculum, CA3, and CA1. In contrast, the receptor-mediated response was maintained in the dentate gyrus and hilus. These results indicate that the age-associated impairment in receptor-mediated PI turnover differs regionally, with a reduction in the subiculum and hippocampus proper that is pronounced relative to the hilus and dentate gyrus.

Aging↗

PubMed2001

Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents.

Neurons and glia within the hippocampus of aged, spatial learning-impaired Long-Evans rats exhibit uniquely altered gene expression profiles, and we have postulated oxidative stress as the basis for this. To test this hypothesis we quantitated the extent of protein and nucleic acid oxidative damage, evaluated the status of mitochondrial DNA integrity, and examined several signaling entities and molecular indicators frequently associated with oxidative stress and gliosis. Immunoblotting demonstrated elevated heme oxygenase-1 in the aged-impaired hippocampus and immunocytochemistry suggested that heme oxygenase-1 is largely cytosolic and at least partly neuronal in nature. In the aged-impaired group, immunoreactivity to 8-hydroxy-2'-deoxyguanosine, an oxidative nucleic acid adduct, was found to be elevated in the dentate gyrus and in area CA1 of the hippocampal formation. Isolated mitochondrial DNA was found to be significantly damaged in the aged-impaired group. In the aged learning-impaired rats only, proteins in a 65-kDa band were found to contain excessive levels of carbonyl residues. Glial activation was examined by in situ hybridization histochemistry to tumor necrosis factor alpha and by immunocytochemistry with OX-6, which detects activated microglia. White matter in aged brains exhibited a modest up-regulation of tumor necrosis factor alpha mRNA and OX-6 immunoreactivity, but the hippocampal formation expressed tumor necrosis factor alpha mRNA equivalent to young animals and few OX-6-positive microglia. The mRNA for manganese-dependent superoxide dismutase, which is elevated in the aged hippocampus, was found preferentially expressed in neurons. We conclude that aged hippocampal neurons appear to be under oxidative stress and this is more severe in the learning-impaired subjects, suggesting a possible basis for age-induced cognitive decline.

Aging↗

PubMed2000

Induction of nitric-oxide synthase and activation of NF-kappaB by interleukin-12 p40 in microglial cells.

Interleukin-12 (IL-12) is composed of two different subunits, p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the central nervous system of patients with multiple sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. However, the mode of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital role in the pathophysiology of MS, the present study was undertaken to explore the role of p40 in the induction of NO production and the expression of iNOS in microglia. Both IL-12 and p40(2), the p40 homodimer, dose-dependently induced the production of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of NO production by the expression of mouse p40 cDNA but not that of the mouse p35 cDNA suggests that the p40 but not the p35 subunit of IL-12 is involved in the expression of iNOS. In addition to BV-2 glial cells, p40(2) also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and p40(2) were unable to induce the production of NO in mouse primary astrocytes. Because activation of NF-kappaB is important for the expression of iNOS, we investigated the effect of p40(2) on the activation of NF-kappaB. Induction of the DNA binding as well as the transcriptional activity of NF-kappaB by p40(2) and inhibition of p40(2)-induced expression of iNOS by SN50, a cell-permeable peptide carrying the nuclear localization sequence of p50 NF-kappaB, but not by SN50M, a nonfunctional peptide mutant, suggests that p40(2) induces the expression of iNOS through the activation of NF-kappaB. This study delineates a novel role of IL-12 p40 in inducing the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Animals↗

PubMed2000

Differential vulnerability of primary cultured cholinergic neurons to nitric oxide excess.

Many neuronal nitric oxide synthase (nNOS)-expressing brain neurons, including some cholinergic populations, are resistant to disease or to certain forms of excitotoxicity. Vulnerability to NO excess of forebrain (medial septal/diagonal band; MS-ACh) and brainstem (pedunculopontine/laterodorsal tegmental nuclei; BS-ACh) cholinergic neurons was compared in E16-E18 primary rat brain cultures. MS-ACh cells were approximately 300-fold more sensitive to the NO donor S-nitro-N-acetyl-D,L-penicillamine (SNAP) than were BS-ACh cells. Most (69%) MS-ACh cells contained nuclear DNA fragments by 2 h after addition of SNAP, while only 21% BS-ACh cells were TUNEL-positive after NO excess. Depletion of glutathione content did not potentiate the effect of SNAP on MS-ACh cells, but sensitized BS-ACh cells to the NO donor. Caffeic acid, a putative NF-kappa B inhibitor, enhanced the toxicity of SNAP to cholinergic neurons in both preparations. Our experiments show that cholinergic neurons in mixed primary cultures from different brain regions possess biochemical differences with respect to their vulnerability to NO excess.

Acetylcholine↗

PubMed2000

Design, synthesis and binding at cloned muscarinic receptors of N-[5-(1'-substituted-acetoxymethyl)-3-oxadiazolyl] and N-[4-(1'-substituted-acetoxymethyl)-2-dioxolanyl] dialkyl amines.

Few muscarinic antagonists differentiate between the M4 and M2 muscarinic receptors. In a structure activity study, aimed at discovering leads for the development of a M4 muscarinic receptor-selective antagonist, we have synthesized and tested at cloned muscarinic receptors the binding of a group of dioxolane- or oxadiazole-dialkyl amines, and compared them to our compound 1, which contains the furan nucleus. Although none of these agents were particularly potent at M4 receptors (Kd values were typically 30-70 nM), furan derivatives (-)1 and (+)1 were significantly more potent at M4 receptors than at M2 receptors (approximately 3- and 4-fold, respectively). The dioxolane derivatives 12b and 12c were more than 10-fold selective for the M4 versus the M2 receptors, while the dioxolane derivative 12e was 15-fold more potent at M4 receptors than for M2 receptors. However, these agents bound to M3 receptors with potencies like that for the M4 receptor, so they are not M4-selective. The M4/M2 relative selectivities of some of our compounds are similar to the better hexahydrosiladifenidol derivatives, and may provide some important structural clues for the development of potent and selective M4 antagonists.

Amines↗

PubMed2000

Retinoic acid-mediated enhancement of the cholinergic/neuronal nitric oxide synthase phenotype of the medial septal SN56 clone: establishment of a nitric oxide-sensitive proapoptotic state.

It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 microM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3.

Acetylcholine↗

PubMed1999

Metabotropic glutamate receptor-mediated hippocampal phosphoinositide turnover is blunted in spatial learning-impaired aged rats.

Maximal phosphoinositide (PI) turnover was examined in the hippocampus of young and aged Long-Evans rats that were behaviorally characterized for spatial learning in the Morris water maze. The type 1 metabotropic glutamate receptor (mGluR) agonist 1S,3R ACPD was used to stimulate PI turnover and to determine the E(MAX) for each rat. Protein levels in hippocampus for type 1 mGluRs, Galphaq11, and phospholipase Cbeta-1 (PLCbeta-1) were also measured by quantitative Western blotting. The results show that PI turnover mediated by the mGluRs was blunted in the aged rats. The magnitude of the decrement in PI turnover was also significantly correlated with age-related spatial memory decline. The decrease in mGluR-mediated PI turnover occurred without changes in the protein level of either the mGluRs or the G-protein coupled to those receptors, Galphaq11. A significant decrease in the immunoreactivity of PLCbeta-1, however, was observed in the hippocampus of aged rats; PLCbeta-1 immunoreactivity was significantly correlated with spatial learning only when the young and aged rats were considered together. The decrement in mGluR-mediated signal transduction in the hippocampus that is related to cognitive impairment in aging may be attributable, at least in part, to a deficiency in the enzyme PLCbeta-1. That deficiency may also contribute to a blunted response in muscarinic stimulation of hippocampal PI turnover that we previously found in this same study population. An age-related alteration in this signal transduction system may provide a functional basis for cognitive decline independent of any loss of neurons in the hippocampus.

Aging↗

PubMed1999

Integrating quantitative and qualitative methods to study multifetal pregnancy reduction.

This study integrates quantitative and qualitative research methods to examine the psychologic repercussions of multifetal pregnancy reduction, a recently developed reproductive technology. Two theoretical vantage points, descriptive psychiatry and psychoanalytic theory, were used to understand the emotional impact of the medical intervention, which involves aborting some but not all of the fetuses in a multifetal pregnancy. Quantitative analysis of diagnostic interviews indicated that women who underwent pregnancy reductions were at no greater risk than controls for developing depressive disorder. Although multifetal pregnancy reduction posed no apparent mental health risk, women experienced it as stressful and distressing. Women's responses were organized and understood via qualitative analyses based on six contemporary psychoanalytic perspectives: drive theory, ego psychology, object relations theory, self-psychology, interpersonal viewpoints, and developmental concepts. Some of the practical and philosophic implications of qualitative and quantitative strategies are considered.

Adaptation, Physiological↗

PubMed1999

Expression of superoxide dismutase messenger RNA in adult rat brain cholinergic neurons.

Superoxide dismutase (SOD) protects cells exposed to an excess of the free radical nitric oxide, by preventing the formation of peroxynitrite. Certain central cholinergic neurons express constitutive nitric oxide synthase (nNOS), and presumably they are at risk from peroxynitrite intoxication. Immunocytochemistry for choline acetyltransferase (ChAT) was combined with in situ hybridization histochemistry (ISHH) to examine whether brain cholinergic populations differ with respect to their expression of the messenger RNA molecules (mRNAs) for the manganese-dependent (Mn-SOD) and copper/zinc-dependent superoxide dismutases (Cu /Zn-SOD). The cholinergic neurons located in the reticular formation of the upper brainstem (the laterodorsal tegmental nucleus [LDTN] and the pedunculopontine nucleus [PPN]) were found to express relatively high levels of Mn-SOD mRNA, whereas cholinergic neurons located in the basal forebrain (substantia innominata [SI], diagonal band [DB], medial septum [MS], and the nucleus basalis magnocellularis [nBM]), and the striatal cholinergic interneurons expressed low to intermediate levels of Mn-SOD mRNA. The rank order of median Mn-SOD mRNA density per cholinergic cell was LDTN > PPN > SI > striatum = nBM = DB > MS. This is similar to the rank order of nNOS mRNA densities in the cholinergic cells in these regions (R = 0.9, p < 0.02). The rank order of Cu/Zn-SOD mRNA levels in cholinergic populations (DB > LDTN = PPN =MS > SI = nBM = striatum) was not correlated with nNOS mRNA (R = 0.29, P > 0.05). Thus, for cholinergic neurons, Mn-SOD may be important for protection from NO-related oxidative stress.

Animals↗

PubMed1998

Indicators of glial activation and brain oxidative stress after intraventricular infusion of endotoxin.

Glial activation and oxidative stress are both consequences of brain aging. To investigate whether glial activation causes oxidative stress or not, the immune activator, lipopolysaccharide (LPS), was intraventricularly injected into the rat brain. The expression of candidate genes were examined by in situ hybridization histochemistry (ISHH) combined with immunohistochemistry for glial markers over a period of time up to 24 h after the LPS injection. The mRNA for glial fibrillary acidic protein (GFAP) was elevated around the injection site by 2 h, and the volume of elevated expression spread to the entire brain after 6 h, with higher levels present in the injected hemisphere. The level of inducible isoform of nitric oxide synthase (i-NOS) mRNA increased in a punctate-like pattern in the region of the injection by 6 h and this response spread to the entire brain after 12 h. These results indicate that the glia are activated for at least 24 h after a single LPS injection. The mRNAs for a heat-shock protein (HSP70) and for the manganese-dependent superoxide dismutase (Mn-SOD) were elevated in the ipsilateral hemisphere as early as 2 h post-injection, but these responses subsided nearly to basal levels by 4 h. These levels of mRNAs for these genes increased again after 6 h of the LPS injection; thus, the earlier increases of the messages appeared to be associated with the survival surgery procedure. With microautoradiographic analysis, scattered OX-42 positive cells expressed i-NOS mRNA after 6 h post-injection, but elevation of Mn-SOD mRNA was not detected in either microglia or astrocytes at any time point examined. The level for Cu/Zn-SOD mRNA did not alter at any time point. The beta-amyloid precursor protein (betaAPP) mRNAs were elevated beginning at 6 h. These results indicate that chronic glial activation leads to a condition of oxidative stress in the brain. The data also suggest that LPS injection could be used to study the effects of chronic glial activation on the survival of neuronal populations that could be at risk from oxidative stress.

Animals↗

PubMed1998

Congener-specific analysis of PCBs in human milk from Kazakhstan.

We have characterized the spectrum of PCB contaminants in human milk samples from 95 women in Kazakhstan using GC/MS congener specific analysis. In these samples, 12 PCBs comprised 83% of total PCB concentration, and were similar to the major PCBs reported in other published human milk studies. By summing concentration levels of 80 PCB congeners in the Kazakhstan samples, a mean total PCB concentration of 368 ng/g fat was obtained. This is lower than levels reported in human milk samples from Western Europe. Six indicator PCBs were summed in Kazakh milk samples; their aggregate value was similar to literature values published for human milk from the former Soviet Union. Using WHO's recent TEF scheme, the mean sum of PCDD/F, coplanar PCB, mono-ortho PCB, and di-ortho PCB TEQ is 42 pg TEQ/g fat.

Benzofurans↗

PubMed1998

Analysis of breast milk to assess exposure to chlorinated contaminants in Kazakstan: high levels of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in agricultural villages of southern Kazakstan.

To assess levels of chlorinated contaminants in breast milk, we measured organochlorine pesticides, polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) in breast milk samples collected in 1994 according to the World Health Organization protocol from 92 donors that were representative of regional populations in southern Kazakstan. High levels (10-120 pg/g fat) of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic of the PCDD/PCDF congeners, were found in breast milk samples from an agricultural region. TCDD was the major contributor (75%) to the international toxicity equivalents of these samples. The same distinctive PCDD/PCDF congener pattern was found in 15 breast milk samples and 4 serum samples collected in 1996 in a follow-up study, and has now been confirmed by three analytical laboratories.

Adolescent↗

PubMed1997

Induction of manganese superoxide dismutase in BV-2 microglial cells.

The regulation of the manganese-dependent superoxide dismutase (Mn-SOD) was studied in immortalized microglial cells (line BV-2). BV-2 cells, activated with lipopolysaccharide (LPS), exhibited an increase in Mn-SOD-like immunoreactivity, that was associated with an accumulation of nitrite in the culture medium and an increase in immunoreactivity for the inducible type of nitric oxide synthase (i-NOS). The i-NOS inhibitor L-N6-(1-iminoethyl)-lysine (NIL, 600 microM) suppressed the nitrite accumulation and the increase in Mn-SOD-like immunoreactivity in activated cells without significant effect on the level of i-NOS-like immunoreactivity. The NO donor sodium nitroprusside dose-dependently increased Mn-SOD-like immunoreactivity in NIL-pretreated BV-2 cells. These results indicate that the induction of Mn-SOD in activated BV-2 cells is mediated in part by NO, or its metabolites.

Animals↗

PubMed1997

mRNA for the m4 muscarinic receptor subtype is expressed in adult rat brain cholinergic neurons.

A number of pharmacological, anatomical, and immunological studies have previously addressed the subtype identity of the hippocampal muscarinic pre-synaptic autoreceptor. A preponderance of findings indicate that it is of the M2 pharmacological type. Both the m2 and m4 molecular subtypes exhibit M2 pharmacology and there are few drugs that differentiate between these receptors. Pharmacological attempts at defining the hippocampal autoreceptor have yielded conflicting results. The basal forebrain is relatively enriched in m2 muscarinic receptor mRNA and protein, and lesions that denervate the hippocampus of its basal forebrain cholinergic input have shown a decrement in m2, but not m4, receptor protein in the hippocampus. Thus, the anatomical data obtained to date tend to support the view that the m2 subtype is expressed as the hippocampal autoreceptor. We have combined in situ hybridization histochemistry (ISHH) with immunocytochemistry to choline acetyltransferase to examine whether mRNA for the m4 subtype of muscarinic receptor is expressed in central cholinergic neurons. The m4 muscarinic mRNA was found at moderate levels in all subdivisions of the cholinergic basal forebrain, including the medial septum/diagonal band complex (MS/DB). The m4 mRNA was also found in striatal cholinergic interneurons, in the cholinergic reticular core of the upper brainstem, and in brainstem cholinergic motor neurons. Muscarinic m4 receptor mRNA was also found in many non-cholinergic cells in the brain. For example, the hippocampal pyramidal neurons, dentate gyrus granule cells, and entorhinal cortical pyramidal neurons express relatively high levels of m4 mRNA, while in the brainstem the dorsal raphe and pontine reticular nuclei express relatively high levels of this mRNA. The finding of m4 mRNA in the MS/DB cholinergic neurons suggests that this receptor protein might be expressed as an autoreceptor in hippocampal cholinergic terminals.

Animals↗

PubMed1997

Immunostimulation protects microglial cells from nitric oxide-mediated apoptosis.

Cellular oxidative stress from excess free radicals can initiate apoptosis in some cell types. Thus, we hypothesize that cells expressing nitric oxide synthase (NOS) will also express anti-oxidative stress mechanisms, in order to prevent self-intoxication by the free radical, nitric oxide (NO). To test this hypothesis, we investigated the vulnerability of microglial cells (BV-2 line) to NO produced by the NO donor sodium nitroprusside. Damage to the cells was measured by an in situ detection method of DNA fragmentation, an indicator of apoptosis. Activation by lipopolysaccharide (LPS) dose-dependently protected BV-2 cells against NO toxicity (up to 67%) while unactivated BV-2 cells were vulnerable to NO. Our results indicate that activation of BV-2 cells induces protection mechanisms against NO toxicity.

Animals↗

PubMed1997

Use of capillary electrophoresis with laser-induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: applications in the cloning of cells.

Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; i.e., basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter-specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c-NOS), and the growth-associated protein GAP-43, three genes known to be expressed in central cholinergic neurons. A "nondestructive" method of screening cultured cells for their expression of c-NOS was established using depolarization with medium containing 50 mM potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E-115 (c-NOS-positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature-sensitive SV-40 transforming activity and neomycin resistance. Cell colonies surviving in G418-containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33 degrees C, differentiated by switching to a low-serum medium and growth at 39 degrees C, and screened for depolarization-induced accumulation of nitrite in the medium. The subset of putative c-NOS-positive clones (about 4%) were then screened for their expression of mRNAs using RT/PCR in combination with CE/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.

Animals↗