Balancing a large scale of responsibility: an interview with R. Milton Johnson.
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Financial Management, Hospital↗
Biomedical subjects
R M Johnson
Publications and source records attributed to R M Johnson.
Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.
A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.
Action Potentials↗
Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene.
To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF. Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription. Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo. The results suggest GDNF is regulated in a complex manner.
Adult↗
Inhibition of anandamide hydrolysis by the enantiomers of ibuprofen, ketorolac, and flurbiprofen.
The endogenous cannabimimetic anandamide is hydrolyzed by a fatty acid amide hydrolase to yield arachidonic acid and ethanolamine. In the present study, the regional distribution of the activity and its sensitivity to inhibition by the enantiomers of ibuprofen, ketorolac, and flurbiprofen has been investigated. The rate of [3H]anandamide hydrolysis was found in both 7-week-old and 90-week-old rats to be in the order hippocampus > cerebral cortex > cerebellum > striatum approximately midbrain, with higher rates of hydrolysis for the 7-week-old rats than for the 90-week-old rats. In whole brain (minus cerebellum), the R(-)-enantiomer of ibuprofen was a mixed-type inhibitor of anandamide hydrolysis and was approximately 2-3 times more potent than the S(+)-enantiomer, IC50 values of 230 and 750 microM, respectively, being found. A similar pattern of inhibition of anandamide hydrolysis was seen when intact C6 rat glioma cells were used. Ketorolac inhibited rat brain anandamide hydrolysis, with IC50 values of 50, 440, and 80 microM being found for the R-, S-, and R,S-forms, respectively. The IC50 value for R-flurbiprofen (60 microM) was similar to the IC50 value for the S-enantiomer (50 microM). These data demonstrate that there is no dramatic enantiomeric selectivity of NSAID compounds as inhibitors of fatty acid amide hydrolase enzyme(s) responsible for the hydrolysis of anandamide. The enantiomers of flurbiprofen and R-ketorolac are the most potent NSAID inhibitors of fatty acid amide hydrolase yet reported.
Aging↗
Differential regulation of glial cell line-derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines.
Human SK-N-AS neuroblastoma and U-87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line-derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24-hr exposure to tumor necrosis factor-alpha (TNFalpha; 10 ng/ml) or interleukin-1beta (IL-1,; 10 ng/ml) induced GDNF release in U-87MG cells, but repressed GDNF release from SK-N-AS cells. Fibroblast growth factors (FGF)-1, -2, and -9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 microM), phorbol 12,13-didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 microM), and vitamin D3 (1 microm) also differentially effected GDNF release from U-87MG and SK-N-AS cells. A result shared by both cell lines, was a two- to threefold increase in GDNF release by db-cAMP (1 mM), or forskolin (10 microM). In general, analysis of steady-state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U-87MG cells but remained static in SK-N-AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous factors, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U-87MG) and neuronal (SK-N-AS) origin.
Brain Neoplasms↗
Increased erythrocyte deformability in fetal erythropoiesis and in erythrocytes deficient in glucose-6-phosphate dehydrogenase and other glycolytic enzymes.
Erythrocyte deformability was determined in more than 500 clinical samples, and was found to be elevated in conditions in which fetal-like red cells are produced: aplastic anemia (3/3 cases), myelodysplastic syndromes, polycythemias, sickle cell anemia during treatment with hydroxyurea, paroxysmal nocturnal hemoglobinuria, and recovery from B12 deficiency. Elevated deformability was observed in neonatal erythrocytes, and during recovery from transient erythroblastopenia of childhood, when fetal-like red cells are known to be produced. Increased deformability appears to be a feature of fetal and fetal-like red cells. Forty-eight cases of enzymatically verified glucose-6-phosphate (G-6-PD) deficiency were also examined. Thirty out of 32 G-6-PD(A-) individuals, including both heterozygotes and hemizygotes, exhibited increased deformability during the steady state. In contrast, G-6-PD(Med) hemizygotes had normal deformability. Increased deformability was also found in G-6-PD(Huron) (n=3), G-6-PD(Wayne) (n=4), triose phosphate isomerase deficiency (n=2), and pyruvate kinase deficiency (n=2). An elevated osmoscan was found in more than 90% of female G-6-PD heterozygotes, affording a simple screening test for heterozygotes. Deformability remained high during hemolytic episodes, when older enzyme deficient cells are removed from the circulation. In four cases of G-6-PD deficiency with normal deformability, evidence for co-existing hereditary spherocytosis was found. The combination of conditions with opposing effects on deformability resulted in nearly normal deformability. Because increased red cell deformability is a feature of fetal erythrocytes, these results suggest that the red cells in many cases of glycolytic enzyme deficiency are fetal-like.
Anemia, Sickle Cell↗
Characterization of the analgesic and anti-inflammatory activities of ketorolac and its enantiomers in the rat.
The marked analgesic efficacy of ketorolac in humans, relative to other nonsteroidal anti-inflammatory drugs (NSAIDs), has lead to speculation as to whether additional non-NSAID mechanism(s) contribute to its analgesic actions. To evaluate this possibility, we characterized (R,S)-ketorolac's pharmacological properties in vivo and in vitro using the nonselective cyclooxygenase (COX) inhibitors [indomethacin (INDO) and diclofenac sodium (DS)] as well as the selective COX-2 inhibitor, celecoxib, as references. The potency of racemic (R,S)-ketorolac was similar in tests of acetic acid-induced writhing, carrageenan-induced paw hyperalgesia, and carrageenan-induced edema formation in rats; ID50 values = 0.24, 0. 29, and 0.08 mg/kg, respectively. (R,S)-ketorolac's actions were stereospecific, with (S)-ketorolac possessing the biological activity of the racemate in the above tests. The analgesic potencies for (R,S)-, (S)-, and (R)-ketorolac, INDO, and DS were highly correlated with their anti-inflammatory potencies, suggesting a common mechanism. (R,S)-ketorolac was significantly more potent than INDO or DS in vivo. Neither difference in relative potency of COX inhibition for (R,S)-ketorolac over INDO and DS nor activity of (S)-ketorolac at a number of other enzymes, channels, or receptors could account for the differences in observed potency. The distribution coefficient for (R,S)-ketorolac was approximately 30-fold less than for DS or INDO, indicating that (R,S)-ketorolac is much less lipophilic than these NSAIDs. Therefore, the physicochemical and pharmacokinetics properties of (R,S)-ketorolac may optimize the concentrations of (S)-ketorolac at its biological target(s), resulting in greater efficacy and potency in vivo.
Acetic Acid↗
Phosphatidylcholine-specific phospholipase inhibitor D609 differentially affects MAP kinases and immediate-early genes in PC12 cells.
The effects of tricyclodecan-9-yl xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipases, on PC12 cells were investigated. D609 repressed nerve growth factor (NGF)-mediated induction of c-fos mRNA with an IC50 approximately 50 microg/ml. Interestingly, maximal c-fos-suppressing doses of D609 did not affect activity of extracellular signal-regulated kinases. Surprisingly, D609 enhanced the extracellular acidification rate of PC12 cells, even in the absence of NGF. D609 alone induced c-jun mRNA with the same potency as it repressed the NGF-induced expression of c-fos. Like NGF, D609 alone induced c-jun even in the presence of dominant-negative Ras. Immediate-early induction of c-jun mRNA by NGF and D609 was abrogated by pretreatment with the kinase inhibitor olomoucine. Jun kinase, which is inhibited by olomoucine, was found to be activated by D609. Thus, D609 might induce c-jun in PC12 cells as a consequence of Jun kinase activation through a Ras-independent pathway. Under the same conditions, D609 repressed NGF-mediated induction of c-fos mRNA.
Animals↗
Regulation of glial cell line-derived neurotrophic factor release from rat C6 glioblastoma cells.
We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, -2, or -9 elevated secreted GDNF protein levels five- to 10-fold. The proinflammatory cytokines interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 nM) activation of protein kinase C, the Ca2+ ionophore A23187 (1 microM), okadaic acid (10 nM) inhibition of type-2A protein phosphatases, nitric oxide donors (1 mM), and H2O2 (1 mM)-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 microM). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.
Animals↗
Hyperplastic changes within the leptomeninges of the rat and monkey in response to chronic intracerebroventricular infusion of nerve growth factor.
Recombinant human nerve growth factor (rhNGF) was delivered for up to 6 months by continuous intracerebroventricular (i.c.v.) infusion to CD (Sprague-Dawley derived) rats and cynomolgus monkeys. Rats (n = 15/sex/group) received doses of 0 (vehicle), 6, 60, or 300 ng/day; monkeys (n = 5/sex/group) received 0, 0.6, 6, or 60 microg/day of rhNGF. Animals tolerated i.c.v. infusion with no behavioral signs attributable to rhNGF. Body weight was transiently decreased in female rats at the highest dose. At the completion of dosing, histological examination in both species revealed an increase in the thickness of the leptomeninges along the ventral and lateral surfaces of the hindbrain and extending over the dorsal aspect of the spinal cord. The change was present to varying degrees at all doses of rhNGF and tended to be more severe at higher doses. At the light microscopic level, the leptomeninges contained layers of well-differentiated, spindle-shaped cells and a plexus of axonal fibers. Cells were immunoreactive for S-100 protein and were associated with an accumulation of Type IV collagen, suggesting Schwann cell origin. Electron microscopy revealed numerous fine caliber axons ensheathed by the presumptive Schwann cells, with myelination of individual axonal segments. These findings suggest that chronic i.c.v. delivery of rhNGF has stimulated axonal sprouting and secondary hyperplasia of Schwann or Schwann-like support cells within the pia-arachnoid.
Animals↗
The TRAM flap for breast reconstruction.
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Blood Transfusion↗
Identification of pre-gamma-globin.
A pre-gamma-globin species was identified by high performance liquid chromatography in platyrrhine primates. Although pre-gamma-globin has not been observed in human hemoglobin, its identification in platyrrhine hemoglobin was facilitated by the functional inactivation of one of the duplicated gamma-globin genes in platyrrhines, which simplified the high performance liquid chromatography elution pattern. Part, but not all, of the pre-gamma was glutathionyl gamma 2-globin, and matrix-assisted laser desorption/ionization mass spectrometry was used to demonstrate that the glutathionyl residue was located on cysteine 93. As this residue is invariant in primates, it is predicted that the formation of glutathionyl gamma-globin will be seen in all primate hemoglobins under appropriate conditions.
Amino Acid Sequence↗
Fetal globin expression in New World monkeys.
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Amino Acid Sequence↗
Developmental abnormalities and age-related neurodegeneration in a mouse model of Down syndrome.
To study the pathogenesis of central nervous system abnormalities in Down syndrome (DS), we have analyzed a new genetic model of DS, the partial trisomy 16 (Ts65Dn) mouse. Ts65Dn mice have an extra copy of the distal aspect of mouse chromosome 16, a segment homologous to human chromosome 21 that contains much of the genetic material responsible for the DS phenotype. Ts65Dn mice show developmental delay during the postnatal period as well as abnormal behaviors in both young and adult animals that may be analogous to mental retardation. Though the Ts65Dn brain is normal on gross examination, there is age-related degeneration of septohippocampal cholinergic neurons and astrocytic hypertrophy, markers of the Alzheimer disease pathology that is present in elderly DS individuals. These findings suggest that Ts65Dn mice may be used to study certain developmental and degenerative abnormalities in the DS brain.
Aging↗
Fetal globin expression in New World monkeys.
Reverse phase chromatography of the globin chains of adult, newborn, and fetal erythrocytes from three species of New World monkeys (Cebus apella, Aotus azarae, and Callithrix jacchus) representing three of the seven platyrrhine clades showed that gamma-globin expression was fetal in these animals. The globins were identified by a combination of chemical sequencing and mass spectrometric analysis. Since gamma-globin expression is fetal in the other major simian branch, the catarrhines, but embryonic in prosimian primates and nonprimate placental mammals, the evolution of fetal recruitment can now be assigned to the period between the simian-prosimian divergence (55 million years ago) and the platyrrhine-catarrhine divergence (35 million years ago). The gamma-globin gene underwent tandem duplication during the same evolutionary epoch, in accord with a model that suggests that the downstream duplicated gamma-gene (gamma2) was free to acquire the mutations necessary for fetal recruitment. Mass spectrometric analysis of tryptic digests of the gamma-globins verified the amino acid sequences deduced from genomic sequencing. Detailed analysis of high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry data showed that gamma2-globin in Cebus was expressed to a far greater extent than gamma1-globin, supporting inferences drawn from a study of the promoter sequences. A "pre-gamma"-globin was observed in C. apella and shown to be primarily the glutathionyl adduct. The other species, A. azarae and C. jacchus, also express only one gamma-globin polypeptide. This work provides biochemical evidence of an evolutionary trend in the platyrrhines to alter the duplicated gamma-globin gene locus so that only one gamma-globin polypeptide is expressed.
Abortion, Veterinary↗
Distribution of radioiodinated recombinant human nerve growth factor in primate brain following intracerebroventricular infusion.
The distribution of radioiodinated recombinant human nerve growth factor ([125I]rhNGF) was evaluated in adult cynomolgus monkeys following unilateral intracerebroventricular (icv) administration. Animals were cannulated into the right ventricle and recovered for 7 days. Monkeys were infused with 1.2 micrograms of [125I]rhNGF or [125I]rhNGF with a 140-fold excess of rhNGF. Twenty-four hours after infusion, animals were anesthetized and transcardially perfused with an aldehyde fixative. Coronal brain sections were processed for quantitative film autoradiography or for choline-acetyltransferase immunohistochemistry and then emulsion dipped. Specific radiolabel was distributed bilaterally and, with equal density, throughout the basal forebrain and was colocalized with choline acetyltransferase-positive neurons. Specific labeling was also present in the superficial ventral cortex. Nonspecific binding was observed surrounding the ventricles and lining blood vessels. These results demonstrate that unilateral icv infusion is an effective approach for delivering NGF to basal forebrain cholinergic neurons in primates and represents a viable drug delivery strategy for the therapeutic use of NGF in Alzheimer's Disease.
Animals↗
Osmotic scan ektacytometry in clinical diagnosis.
PURPOSE: The use of red blood cell deformability measurements in the diagnosis of hemolytic anemia is reviewed. PATIENTS AND METHODS: Results from 500 individuals are discussed. Erythrocytes were characterized by the automated measurement of cell deformability as the tonicity of the medium is varied (osmotic scan ektacytometry). RESULTS: The measurement yields a reliable identification of hemolytic anemias caused by deficiencies or abnormalities in erythrocyte structural proteins. In addition, it can, with good reliability, signal the presence of a glycolytic enzyme deficiency. The scan is complete in 15 min, and can therefore give a rapid indication of the type of hemolytic anemia. The osmotic scans of other hemolytic anemias are also discussed. CONCLUSION: The analysis of red blood cell deformability can offer a valuable addition to diagnostic methods in hemolytic anemia.
Anemia, Hemolytic↗
Dose-response comparison of recombinant human nerve growth factor and recombinant human basic fibroblast growth factor in the fimbria fornix model of acute cholinergic degeneration.
Both nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) have been proposed for the treatment of Alzheimer's disease. This study describes a comparative, dose-response analysis of recombinant human (rh)NGF and rhbFGF in a rat unilateral fimbria-fornix model of acute cholinergic neuronal degeneration. Doses for rhNGF were 0.6, 6, 60, 600 and 1,800 ng/rat/day and for rhbFGF were 600, 1,800, 3,000 and 6,000 ng/rat/day, delivered for 4 weeks. The number of surviving septal cholinergic neurons was evaluated using ChAT immunohistochemistry. In control animals, the number of ChAT-positive neurons remaining on the lesioned side was between 22 and 18% compared to the non-lesioned side. Infusion with either neurotrophic factor increased the number of ChAT-positive neurons on the lesioned side in a dose-dependent manner. The maximal response to rhbFGF peaked at 3,000 ng/rat/day with a cell savings of 47%. However, there was evidence of neuropathological changes associated with rhbFGF. In contrast, rhNGF produced a maximal response with an infusion of 600 ng rhNGF/rat/day and a cell savings of 70% and no evidence of neuropathology, indicating that rhNGF was better tolerated and more efficacious than rhbFGF.
Acetylcholinesterase↗