Biomedical subjects

C D Earl

Publications and source records attributed to C D Earl.

At least 19 recordsLinked to original sources

PubMed2004

Slow N-acetyltransferase 2 status leads to enhanced intrastriatal dopamine depletion in 6-hydroxydopamine-lesioned rats.

We previously reported an association between the N-acetyltransferase 2 (NAT2) slow acetylator status and Parkinson's disease (PD). We have now investigated the possible functional relevance of this association by treating Fischer 344 (F344) rapid and Wistar-Kyoto (WKY) slow NAT2 acetylator rat strains with the neurotoxin 6-hydroxydopamine (6-OHDA). Intrastriatal treatment with either 10 or 20 microg of 6-OHDA lead to a significantly greater reduction of striatal dopamine concentrations in the WKY slow acetylator rat strain than in the F344 rapid acetylator rat strain (P < 0.004), reflecting a more marked degree of dopaminergic denervation. Nigral dopaminergic cell counts were also lower in the WKY rats, but this difference failed to reach statistical significance, suggesting that slow acetylation is especially deleterious at the level of striatal nerve endings.

Animals↗

PubMed2001

The development of dopamine overflow from foetal nigral grafts in the intact rat striatum and their influence on contralateral striatal dopamine overflow.

In this study, cell suspensions of foetal rat ventral mesencephalic dopaminergic tissue were grafted to the intact (non-lesioned) striatum of adult rats. Differential pulse voltammetry at carbon-fibre micro electrodes (12 microm diameter) was employed to first, monitor the development of dopamine overflow over a 20 week period within the grafts and secondly, their influence on contralateral striatal dopamine overflow. At 8 and 20 weeks, animals were pre-treated with pargyline and both striata were monitored for dopamine overflow for 90 min following d-amphetamine administration. Amphetamine led to a significant increase in dopamine overflow in both the grafted striatum and the contralateral striatum. The time course of dopamine overflow in both the grafted striatum and the striatum contralateral to the graft was similar in all groups of animals. Although the actual concentration of dopamine measured in 20 week old grafts was more (approximately 21%) than that measured in 8 week old grafts, there was no significant difference between the two time points. The concentration of dopamine measured in the striatum contralateral to 8 week old grafts was significantly lower (approximately 43%) than that measured in the striatum of a normal control rats. There was no significant difference between the concentration of dopamine measured in the striatum contralateral to 20 week old grafts and normal control rats. In conclusion, dopamine overflow from a ventral mesencephalic graft does not change significantly between 8 and 20 weeks following grafting. However, the grafted tissue causes a decrease of d-amphetamine-induced dopamine overflow in the contralateral side 8 weeks following grafting, which is restored 12 weeks later.

Animals↗

PubMed2000

Enzymatic mutation detection (EMD) of novel mutations (R565X and R1523X) in the FBN1 gene of patients with Marfan syndrome using T4 endonuclease VII.

The Enzymatic Mutation Detection (EMDtrade mark) method is a streamlined and improved version of the original Enzymatic Cleavage of Mismatch (EMC) method. EMD is a fully homogeneous, rapid four step procedure that allows for detection and localization of mismatched or unmatched nucleotides within heteroduplex DNA. To test the utility of EMD for use in the screening of large and complex genes, the fibrillin 1 (FBN1) gene was scanned in a cohort of six patients diagnosed with connective tissue disorders. Four of the six patients were diagnosed with classic Marfan syndrome (MFS). The results were compared with a previous MDEtrade mark scanning of the same patient cohort. Two causative mutations, R565X and R1523X, were detected by EMD that were not detected by MDE. In both cases, the mutation resulted in premature termination of translation. In addition, several polymorphisms were detected by the enzymatic approach that failed detection by heteroduplex analysis. We propose that the EMD method is a sensitive and rapid approach to mutation detection in large genes such as FBN1.

Adult↗

PubMed2000

Systemic treatment with GM1 ganglioside improves survival and function of cryopreserved embryonic midbrain grafted to the 6-hydroxydopamine-lesioned rat striatum.

Cryopreservation may allow long-term storage of embryonic ventral mesencephalon (VM) for neural transplantation. We investigated whether the ganglioside GM1 or the lazaroid tirilazad mesylate (U-74006F) could improve survival of grafts derived from cryopreserved VM in a rat model of Parkinson's disease. VM was dissected from rat embryos (E14-E15), frozen and stored in liquid nitrogen under controlled conditions, thawed, dissociated, and then grafted into the 6-hydroxydopamine-lesioned rat striatum. In Experiment I, VM fragments were exposed in vitro either to GM1 (100 microM) or to lazaroid (0.3 microM) during all preparative steps. In Experiment II, rats receiving GM1-pretreated VM were, in addition, treated systematically with GM1 (30 mg/kg) daily for 3.5 weeks. Rats grafted with untreated cryopreserved or fresh VM were used as controls, respectively. Rats receiving fresh VM control grafts showed complete recovery from lesion-induced rotations after 6 weeks whereas rats grafted with cryopreserved VM (untreated or pretreated) did not recover. Cryografts contained significantly less (18%, control; 23%, GM1; and 12%, lazaroid) tyrosine hydroxylase-positive cells compared to fresh grafts (1415 +/- 153; mean +/- SEM). Graft volume was also significantly smaller after cryopreservation. In contrast, with additional systemic GM1 treatment cryografts contained almost the same number of tyrosine hydroxylase-positive cells (376 +/- 85) as fresh grafts (404 +/- 56), which was significantly more than that of untreated cryografts (147 +/- 20), showed a significantly larger volume (0.15 mm(3)) compared to that of untreated grafts (0.08 mm(3)) (fresh controls, 0.19 mm(3)), and induced significant and complete functional recovery in the rotation test. In conclusion, systemic treatment of rats with GM1 improved the low survival and functional inefficacy of grafts derived from cryopreserved VM whereas tissue pretreatment alone with either GM1 or lazaroid was not effective.

Animals↗

PubMed2000

The protective effects of PBN against MPTP toxicity are independent of hydroxyl radical trapping.

To study the mechanism of the protective effect of the spin-trapping agent alpha-phenyl-N-tert-butyl nitrone (PBN) against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity hydroxyl radicals and functional parameters of neuroprotection were determined. C57BL/6 mice received PBN (100 mg/kg IP) over a time period of 15 days and on day 8 MPTP (40 mg/kg SC). On day 15 striatal levels of dopamine, serotonin, and metabolites were analyzed. For radical determination mice received a single injection of salicylic acid (SA) (100 mg/kg IP) in the time period of 0.5 h before to 72 h after MPTP injection. In vivo maximum hydroxyl radical levels indicated by 2,3-dihydroxybenzoic acid/SA ratios were obtained 4 h after MPTP injection, and were not affected by PBN treatment. However, the MPTP-induced mortality, reduction of locomotor activity, continuous loss of body weight, and striatal dopamine depletion were significantly less pronounced in PBN-treated animals. These results elucidate the time course of hydroxyl free radical formation in MPTP toxicity. PBN improved the functional parameters of neuroprotection against MPTP toxicity, but there is no evidence for hydroxyl radical scavenging properties to this effect.

Animals↗

PubMed1999

Salicylate protects against MPTP-induced impairments in dopaminergic neurotransmission at the striatal and nigral level in mice.

The analgesic and anti-inflammatory drug sodium salicylate was studied for its potential protective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. C 57BL/6 mice were treated with a single dose of sodium salicylate (50 mg/kg or 100 mg/kg i.p.) or saline immediately before injection of MPTP (30 mg/kg or 40 mg/kg s.c.) or saline. Analysis of striatal dopamine and metabolites as well as immunostaining for tyrosine hydroxylase of nigral sections was performed 7 days after MPTP treatment. MPTP (30 mg/kg) led to a strong decrease in striatal dopamine levels (1.87+/-0.27 ng/mg) compared to saline-treated controls (15.72+/-0.78 ng/mg), which was significantly attenuated by sodium salicylate 50 mg/kg and 100 mg/kg (5.59+/-0.56 ng/mg and 8.64+/-0.89 ng/mg, respectively). Remarkably, the MPTP-induced loss of tyrosine hydroxylase immunoreactivity in nigral cell bodies was nearly completely prevented by the higher dose of sodium salicylate. Furthermore, salicylate demonstrated radical scavenging effects in an in vitro Fenton system indicated by HPLC determination of the dihydroxylated reaction products of salicylate, namely, 2,3- and 2,5-dihydroxybenzoic acid. The protective effects of salicylate against reversible or irreversible impairments in dopaminergic neurotransmission after MPTP treatment may be related to its radical scavenging properties and other mechanisms which need to be clarified.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1998

Pharmacological characterisation of dopamine overflow in the striatum of the normal and MPTP-treated common marmoset, studied in vivo using fast cyclic voltammetry, nomifensine and sulpiride.

The in vivo measurement of electrically-evoked dopamine overflow was measured for the first time in the striatum of control and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets using fast cyclic voltammetry at untreated carbon-fibre microelectrodes, (7 microm, o.d.). The identity of dopamine was confirmed using electrochemical, pharmacological and histological criteria and complied with rat data from earlier studies. Dopamine overflow depended on the intensity, number of pulses, and frequency of the applied stimuli. Maximum dopamine overflow occurred using 1.0-2.0 mA, 200 micros pulse width, 150-200 pulses at 80-120 Hz stimulation of the medial forebrain bundle. Evoked dopamine overflow in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated marmosets showed a similar electrochemical and pharmacological profile compared to healthy controls, albeit the concentration detected was significantly reduced. The catecholamine uptake blocker, nomifensine, significantly increased the dopamine signal in control marmosets. However, in contrast, nomifensine had no significant effect on evoked dopamine overflow in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated marmosets. Apart from demonstrating that fast cyclic voltammetry with electrical stimulation can be reliably used to monitor dopamine overflow within the primate brain, our results describe for the first time the technical prerequisites for the fast cyclic voltammetric technique in the non-human primate brain.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1998

Effects of nicotine on hydroxyl free radical formation in vitro and on MPTP-induced neurotoxicity in vivo.

Parkinson's disease (PD) is one of the most frequent disorders of the basal ganglia. From epidemiological studies there is a controversial discussion on the question whether tobacco smoking is correlated with a decreased incidence of PD. The present study aimed to elucidate the role of nicotine and its potential neuroprotective effects in a rodent model of PD. These effects may be related to an altered hydroxyl radical formation; this possibility was studied in vitro. Nicotine and alpha-phenyl-N-tert-butyl nitrone (PBN) were examined in a cell-free in vitro Fenton system (Fe3+/EDTA + H2O2) for their radical scavenging properties using the salicylate trapping method. Salicylic acid (0.5 mM) was incubated in the presence and absence of nicotine or PBN and the main products of the reaction of hydroxyl radicals with salicylic acid, namely 2,3- and 2,5-dihydroxybenzoic acid, were immediately determined using HPLC in combination with electrochemical detection. Nicotine and PBN were both able to significantly reduce hydroxyl radical levels at concentrations of 1, 2.5 and 5 mM. Interestingly, at 5 mM nicotine was able to reduce hydroxyl radical levels significantly more than the radical scavenger PBN (5 mM). To investigate the in vivo effects of nicotine, male C57BL/6 mice were used in the MPTP mouse model of PD. Nicotine (0.1 or 0.4 mg/kg s.c.) was administered twice daily for a period of 14 days. On day 8 a single injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg s.c.) was given as well as an enhanced protocol of nicotine treatment (0.1 or 0.4 mg/kg s.c., 30 min before MPTP and 30, 90, 210, 330, 450, 570 min after MPTP) for a total of seven injections of nicotine. High dosage nicotine treatment significantly increased the MPTP-induced loss of body weight and resulted in a significantly decreased striatal dopamine content and an increased dopamine turnover in comparison with the MPTP-treated controls at day 15. However, the lower dosage of nicotine did not significantly alleviate the MPTP-induced effects, although some parameters showed a slight tendency in this direction. These results demonstrate that in vitro nicotine has radical scavenging properties which might suggest neuroprotective effects. In vivo experiments with nicotine, however, showed that a low dosage of nicotine did not alleviate the MPTP-induced dopamine depletion, but a large dosage even enhanced it.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1998

Automated fluorescent analysis procedure for enzymatic mutation detection.

The Enzymatic Mutation Detection (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism 377 DNA Sequencer, that improves on earlier methods (1, 2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.

Animals↗

PubMed1997

Effects of graft pooling of foetal rat and mouse tissue and immunosuppression in the 6-hydroxydopamine rat model of Parkinson's disease.

We employed intracerebral co-transplantation of foetal xenogeneic striatal mouse tissue and allogeneic rat substantia nigra into the adult rat brain to elucidate the effects of xenogeneic mouse graft on the function and survival of an allogeneic rat graft in 6-hydroxydopamine lesioned Sprague-Dawley rats. Foetal mouse striatum (STR) and rat substantia nigra (VM) were transplanted as non-pooled separate deposits or a pooled cell suspension with or without cyclosporin A (Cy A). Immunosuppressed recipients of pooled rat and mouse co-grafts showed a significantly better compensation of amphetamine-induced rotational behaviour compared with non-immunosuppressed animals with pooled rat and mouse co-grafts 3 and 6 weeks post-grafting. Tyrosine hydroxylase (TH) immunohistochemistry revealed a non-significant reduction in survival in pooled (1806.3+/-367.5 cells) rat and mouse co-transplants without immunosuppression compared with immunosuppressed pooled (3383.3+/-732.7 cells) animals with allo- and xenogeneic tissue and controls (3506.4+/-839.3 cells). Graft volumes were significantly reduced in pooled transplants without immunosuppression (0.1+/-0.026 mm3; ANOVA post-hoc Scheffe F-test, P<0.0001) compared with immunosuppressed recipients (0.7+/-0.1 mm3) and controls (0.6+/-0.1 mm3). In non-pooled allo- and xenogeneic grafts without immunosuppression the survival rate of the TH-immunoreactive cells and graft volumes were reduced (2359.3+/-479.5 cells; 0.2+/-0.043 mm3) compared with immunosuppressed animals (2927.3+/-946.6 cells; 0.6+/-0.2 mm3) and controls (2701.1+/-693.8 cells; 0.3+/-0.1 mm3) without reaching a level of significance. Rejection of mouse tissue was observed in all non-immunosuppressed recipients. In summary: (i) continued immunosuppression yielded significant beneficial effects on function and beneficial effects on survival of pooled grafts with an immunogenetic disparity; (ii) the rejection of a xenogeneic graft component may compromise survival and function of other, allogeneic graft components; and (iii) transplantation of non-pooled allo- and xenogeneic tissues may result in a better survival of the graft compared with pooled cell suspensions.

Animals↗

PubMed1997

Degeneration of pre-labelled nigral neurons induced by intrastriatal 6-hydroxydopamine in the rat: behavioural and biochemical changes and pretreatment with the calcium-entry blocker nimodipine.

Intrastriatal application of 6-hydroxydopamine (6-OHDA) initiates a delayed and progressive loss of nigral dopaminergic neurons and therefore may better resemble the slowly developing neuropathology of Parkinson's disease. We investigated the anatomical, behavioural and biochemical consequences of intrastriatal 6-OHDA after prior labelling of nigral dopaminergic neurons in rats and whether the dihydropyridine L-type calcium channel blocker nimodipine protected from the induced deficits. Adult rats received bilateral intrastriatal injections of the retrograde fluorescence tracer fluorogold and nimodipine (n=12) or placebo (n=9) pellets implanted subcutaneously. One week later all rats were injected unilaterally with 6-OHDA (20 microg) at the same intrastriatal site. Placebo-treated rats displayed relatively few d-amphetamine-induced ipsilateral net rotations (R) (1.3+/-1.4 R/min; mean+/-SEM) 1 week after the lesion with a slight but non-significant decline thereafter (after 2, 3 and 4 weeks). In nimodipine-treated rats the rotation behaviour after 1 week was more prominent (3.5+/-0.8 R/min; mean+/-SEM) with a similar slight decline until week 4. Fluorescent and immunocytochemical analysis of the midbrain after 4 weeks revealed a 35% and 39% loss of tyrosine hydroxylase positive cells and a 62% and 56% (placebo and nimodipine, respectively) loss of fluorogold-labelled cells in the ipsilateral substantia nigra pars compacta. Striatal dopamine levels were reduced to 47% (placebo) and 43% (nimodipine) of the control side and the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid to about 50%. Pretreatment with nimodipine failed to antagonize or to ameliorate any of the lesion-induced deficits. We conclude that pretreatment with 80 mg nimodipine pellets does not prevent nigrostriatal damage induced by intrastriatal 6-OHDA.

Animals↗

PubMed1996

Foetal ventral mesencephalic cell suspension grafts to the 6-hydroxydopamine-lesioned rat reduce the rate of dopamine uptake in the contralateral striatum.

The present study employed fast cyclic voltammetry, at carbon-fibre microelectrodes, to monitor and compare the rate of dopamine uptake in the rat striatum contralateral to (a) the 6-hydroxydopamine (6-OHDA)-lesioned/grafted striatum and (b) the 6-OHDA-lesioned/ sham grafted striatum. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted striatum of unilaterally 6-OHDA-lesioned rats. Six weeks after grafting, animals with functional, mature grafts were monitored for dopamine elimination in the contralateral striatum following electrical stimulation of the median forebrain bundle, before and after treatment with the dopamine uptake inhibitor GBR 12909. Compared to animals with sham grafts, amphetamine-amplified rotational behaviour was significantly reduced in animals with grafts of foetal ventral mesencephalic tissue. Fast cyclic voltammetric measurements followed by evaluation with the aid of a kinetic model revealed that in grafted animals, the rate of dopamine uptake via the high affinity uptake mechanism, following treatment with GBR 12909, was significantly reduced when compared to sham grafted animals.

Amphetamine↗

PubMed1996

Foetal nigral cell suspension grafts influence dopamine release in the non-grafted side in the 6-hydroxydopamine rat model of Parkinson's disease: in vivo voltammetric data.

The present study employed differential-pulse voltammetry to assess the influence of foetal ventral mesencephalic grafts on dopamine overflow in the contralateral caudate putamen of the 6-hydroxydopamine rat model of Parkinson's disease. The experimental design involved measurements of dopamine overflow in the grafted and contralateral striatum. Control measurements of dopamine overflow were performed in 6-hydroxydopamine-lesioned rats only and the caudate putamen of normal control rats. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted caudate putamen of unilaterally 6-hydroxydopamine-lesioned rats. At 6 weeks, animals with functional, mature grafts (as assessed by amphetamine-amplified behavioural asymmetry), were pretreated with pargyline (75 mg/kg i.p.), and both striatal sides were monitored for dopamine overflow for 90 min following amphetamine sulphate administration (5 mg/kg i.p.). The time course of dopamine overflow inside the graft was similar to that in the contralateral caudate putamen of the same animal, the normal control animal and the contralateral caudate putamen of 6-hydroxydopamine-lesioned animals. However, in grafted animals the mean dopamine overflow detected in the contralateral caudate putamen was approximately 34% lower than the concentration of dopamine detected in the contralateral caudate putamen of 6-hydroxydopamine-lesioned control animals and approximately 39% lower than the concentration of dopamine detected in the caudate putamen of the normal control animal. There was no statistical difference in the concentration of amphetamine-induced dopamine overflow between the caudate putamen contralateral to the 6-hydroxydopamine lesion and the caudate putamen of the normal control animal. These data suggest that intrastriatal foetal ventral mesencephalic suspension grafts reduce amphetamine-induced dopamine release in the contralateral non-grafted caudate putamen.

Amphetamine↗

PubMed1995

Stimulus-evoked dopamine overflow in the rat nucleus accumbens is decreased following chronic haloperidol administration: an in vivo voltammetric study.

Fast cyclic voltammetry was used to investigate the effects of chronic haloperidol (HAL) treatment on electrically evoked dopamine (DA) overflow in the nucleus accumbens of the anaesthetized rat in vivo. Evoked DA efflux was significantly reduced in rats treated with 1.0 mg/kg per day HAL for 21 days. In rats treated with 0.5 mg/kg per day, evoked DA overflow was reduced, but did not differ significantly from control values. In untreated animals, injection of a single dose of HAL resulted in a significant increase in the DA overflow evoked by subsequent stimulus trains. In contrast, this HAL challenge did not produce a significant enhancement in evoked DA overflow in any of the HAL-treated groups. These results are consistent with the previous reports that basal DA release is reduced after chronic HAL treatment, and show for the first time that chronic HAL administration decreases stimulus-evoked DA overflow in the rat nucleus accumbens in vivo.

Animals↗

PubMed1995

[Neuronal transplantation in animal models of Parkinson disease: in vivo voltammetry, tissue cryopreservation and immunology].

Neuronal transplantation of dopaminergic embryonic ventral mesencephalic cells aims at replacing the lost striatal dopamine in Parkinson's disease. Functional effects of intrastriatal ventral mesencephalic transplants are reflected in improvements of bradykinesia and rigidity. However, a more widespread clinical application critically depends on further technical refinements, specifically in the area of improved survival of the grafted ventral mesencephalic dopamine neurones. This review highlights some of the progress made in the field of ventral mesencephalic transplantation in animal models of Parkinson's disease and also discusses issues and results obtained by our own research group in Munich. Thus we studied whether cryo- or neuroprotective additives during cryopreservation of ventral mesencephalic tissue can improve survival of such tissue/cells following transplantation. Concerning immunological aspects we studied the effects of pooled or co-transplanted embryonic tissue on survival and function of grafts and whether immunosuppression with cyclosporine A has beneficial effects. Possible neurotoxic effects of cyclosporine A were considered in a separate study. Finally, we established in vivo voltammetry to measure dopamine release from the graft and effects of the grafts on related parts of the dopaminergic system in the living animal. In the clinic we employed guidelines for preoperative patient evaluation and respective tests are described.

Animals↗

PubMed1995

Neuronal transplantation and neurotrophic factors in the treatment of Parkinson's disease--update February 1995.

This review focuses on two restorative strategies against Parkinson's disease: (1) intrastriatal implantation of fetal dopamine producing cells and (2) application of neurotrophic factors. (1) Neuronal transplantation of dopaminergic embryonic ventral mesencephalic cells aims at compensating the striatal dopamine deficit in Parkinson's disease. Clinically, dopaminergic ventral mesencephalic grafts have been demonstrated to ameliorate rigidity, bradykinesia, and efficacy of L-DOPA therapy, for instance reduction of L-DOPA-induced dyskinesias and ON-OFF-fluctuations. Future experimental refinements predominantly concern the improvement of survival of the transplanted fetal dopaminergic neurons and the use of genetically modified cells. (2) Neurotrophic factor therapy relates to the prevention and/or restoration of the progressing nigral cell loss in Parkinson's disease. In this regard special emphasis is put on the newly characterized glial cell line-derived neurotrophic factor (GDNF). More recent results and perspectives of this concept are discussed.

Clinical Trials as Topic↗

PubMed1993

The effects of exogenous nerve growth factor on foetal rat adrenal cells in culture.

Foetal rat adrenal cells, identified as chromaffin cells, maintained in culture in the presence of exogenous NGF, express a neuronal phenotype. Once this neuronal phenotype has been established bundles of outgrowing neurites commonly form links with adjacent reaggregates, and also develop synaptic contacts with the cell bodies of chromaffin cells in the adjacent reaggregates. Subsequent withdrawal of exogenous NGF from the culture medium did not result in the chromaffin cells losing their neuronal phenotype if this had been established before NGF was withdrawn. Tyrosine hydroxylase immunocytochemistry revealed that all neurites developed in culture were positive for the enzyme including those in cultures only transiently exposed to exogenous NGF. These results suggest that exogenous NGF may be essential only until the chromaffin cells develop neurites which make contact with appropriate target cells and that once these links, however immature, have been established, neurites are retained for many weeks in the absence of exogenous NGF.

Adrenal Glands↗

PubMed1987

Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.

Amino Acid Sequence↗