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D K Anderson

Publications and source records attributed to D K Anderson.

At least 19 recordsLinked to original sources
PubMed1995

Regulation of the Caulobacter crescentus rpoN gene and function of the purified sigma 54 in flagellar gene transcription.

The sequential transcription of flagellar (fla) genes in the Caulobacter crescentus cell cycle is controlled by the organization of these genes in a regulatory hierarchy of four levels (I-IV). Level III and level IV genes at the bottom of the hierarchy are dependent on level II genes and are transcribed late in the cell cycle from sigma 54-dependent promoters. To study the regulation of genes at levels III and IV, we have isolated and sequenced the rpoN gene in order to analyze its expression, purified the rpoN gene product, and examined the role of the RpoN protein in initiation of transcription from sigma 54-dependent promoters. We report here epistasis experiments that show rpoN is required for transcription of level III genes, but that the expression of the rpoN gene itself is not dependent on any of the fla genes examined; these results place rpoN at level II near the top of the hierarchy. Consistent with this conclusion were nuclease S1 assays that mapped the rpoN transcription start site and identified a sequence centered at -24, GTTA/TACCA/TT, which is similar to the core consensus sequence of the level IIB fliF, fliL, and fliQ promoters. We purified the full-length rpoN gene product to near homogeneity and demonstrated that the RpoN protein is required for transcription from the well-characterized sigma 54-dependent glnAp2 promoter of Escherichia coli and specifically recognizes the level III flbG gene promoter of C. crescentus. These last results confirm that rpoN encodes the C. crescentus sigma 54 factor and opens the way for the biochemical analysis of transcriptional regulation of level III and IV fla genes.

Amino Acid Sequence↗
PubMed1995

Dynamic assessment of intraspinal neural graft survival using magnetic resonance imaging.

Although previous work has demonstrated the usefulness of magnetic resonance imaging (MRI) for visualizing intraspinal transplants in vivo, the degree to which MRI can differentiate viable fetal neural tissue from evolving spinal cord pathology has not been investigated. Thus, the present study assessed whether MRI performed at earlier postgrafting intervals (0-20 weeks) could document the survival of fetal neural transplants in the injured cat spinal cord. Twelve adult female cats received a hemisection injury at the L1 level, followed immediately by implantation of either embryonic cat spinal cord or neocortex into the cavity. The spinal cords of three control animals were hemisected but received no transplant. Each animal was subsequently imaged at 4 and 8 weeks postoperative. Selected animals from each group were also studied at additional time points ranging from immediately postoperative to 20 weeks. Multislice T2-weighted and intermediate T1-weighted spin-echo images of the lesion or graft site were obtained. Correlative postmortem histological analyses revealed viable donor tissue in 6 of 12 transplant recipients. Spinal cords from the remaining hosts and the control animals all contained cysts at the surgical site that were devoid of donor neural tissue. The graft sites with viable tissue tended to exhibit a slightly hyperintense signal on both intermediate T1-weighted (T1WI) and T2-weighted images (T2WI) throughout the entire experiment. Control cats and cats with failed transplants also were slightly bright on T1WI, but were very hyperintense on T2WI.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Measurement of vascular permeability in spinal cord using Evans Blue spectrophotometry and correction for turbidity.

Vascular permeability can be visualized by Evans Blue (EB) extravasation and quantified by spectrophotometry after formamide extraction of the tissue. However, formamide extracts show significant turbidity, which may contribute to the total optical density at the wavelength of measurement (e.g., 620 lambda). We developed a simple method for estimating the component of the total optical density of a dyed specimen contributed by turbidity. Our method, which uses a determination of turbidity made at another point of the light spectrum (740 lambda), was more precise than two other EB quantification techniques. We therefore recommend it for individual correction of formamide extracts of spinal cord specimens. The application of this technique to the brain remains to be determined.

Animals↗
PubMed1994

Methylprednisolone and membrane properties of primary cultures of mouse spinal cord.

The present study attempts to define the capacity of methylprednisolone sodium succinate (MP) to protect neuronal membranes against a free radical challenge in primary cultures of fetal mouse spinal cord. Incubation of these cultures with MP significantly increased the Na+,K(+)-ATPase activity, an effect that was blocked by the RNA synthesis inhibitor, actinomysin D and the protein synthesis inhibitor, cycloheximide, suggesting an induction of protein synthesis by MP. In contrast, incubation with FeCl2 for 1 or 2 h significantly inhibited Na+,K(+)-ATPase activity and elevated the levels of thiobarbituric acid-reactive substances (TBARS). Pretreatment with MP prevented the rise in TBARS and partially prevented the decrease in Na+,K(+)-ATPase activity for the first hour of FeCl2 incubation, an effect that was lost during the second hour. A second dose of MP after the first hour of incubation with FeCl2 partially restored Na+,K(+)-ATPase activity and reduced TBARS levels after the second hour of exposure to FeCl2. Co-incubation of MP with cycloheximide completely prevented the decrease in Na+,K(+)-ATPase activity seen after a 2-h incubation with FeCl2 and eliminated the need for a second dose of MP after the first hour of incubation with FeCl2. These findings suggest a capacity for rapid protein induction and antioxidant activity for MP in vitro.

Animals↗
PubMed1994

Excitotoxicity of glutamate and four analogs in primary spinal cord cell cultures.

Continuous glutamate exposure produced widespread neuronal damage in mixed whole dissociated murine spinal cord cell cultures. Ethidium bromide and acridine orange staining revealed that a 24 h glutamate exposure produced nearly 98% neuronal cell death but the underlying glia were spared. Continuous exposure to glutamate, N-methyl-D-aspartate (NMDA), kainate and quisqualate produced time-dependent and dose-dependent cell death as measured by the assay of lactate dehydrogenase activity in the cell culture media. Glutamate (500 microM), NMDA (100 microM) and kainate (500 microM) were equally neurotoxic. In contrast, quisqualate (100 microM) was only partially neurotoxic compared to the other glutamate analogs. The neurotoxicity of glutamate was blocked by the NMDA antagonist, MK-801. The neurotoxicity of kainate and quisqualate was blocked with the non-NMDA antagonist CNQX. Continuous exposure to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) was not neurotoxic, even at concentrations up to 1 mM.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1994

Workshop on intraspinal transplantation and clinical application.

The following general conclusions were reached at the workshop: 1. Laboratory studies suggest a potential benefit of cellular transplant therapy for SCI. 2. Some evidence supporting the safety of human fetal transplants is available from clinical studies of transplants in Parkinson's disease and SCI. 3. Assessment criteria and methodology are available, including imaging approaches, validated neurologic scoring systems, detailed electrophysiologic studies of conduction and spinal cord reflexes, and functional scoring approaches. 4. More controlled animal studies are needed (a) to demonstrate efficacy and to evaluate the necessity for immunosuppressive therapy and the overall safety of intraspinal transplantation, (b) to obtain more supporting evidence (e.g., electrophysiologic, histopathologic, MRI, molecular) that would provide insights into ways that transplanted tissue could mediate function, (c) to provide guidance for the procurement, harvesting, preparation, storage, and other logistics related to the use of human cells for transplantation into the spinal cord, (d) to define more thoroughly the cell type(s) that would be most likely to have benefit and the conditions that affect their viability, migration, gene expressions, and proliferation after transplantation, (e) to determine the most optimal time after injury for transplantation, and (f) to clarify patient selection characteristics that might optimize success (i.e., complete vs incomplete injuries, spinal level involved, age of recipient).

Animals↗
PubMed1994

Effect of remacemide hydrochloride on subarachnoid hemorrhage-induced vasospasm in rabbits.

The purpose of this study was to assess the role of an excitatory amino acid (EAA) receptor antagonist (remacemide hydrochloride) in a rabbit model of subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. Cerebral angiograms were performed on 22 rabbits pre-SAH and 72 h post-SAH: 6 rabbits received an injection of mock cerebrospinal fluid (1 ml/kg) into the cisterna magna (group I, the control group); 6 rabbits were subjected to SAH but received no treatment (group II); autologous blood (1 ml/kg) from the central ear artery was injected into the cisterna magna of these rabbits; 6 rabbits were subjected to SAH (1 ml/kg) and treated with intraperitoneal (IP) bolus injections of remacemide hydrochloride (15 mg/kg) every 12 h beginning 30 minutes after SAH (group III); and 4 rabbits were not subjected to SAH but received IP bolus injections of remacemide hydrochloride every 12 h (group IV). Digital subtraction angiography was used to measure the diameter of the basilar artery. At 72 h post-SAH, vasospasm was evident in all untreated rabbits. The diameter of the basilar artery was reduced significantly below pre-SAH levels by 35.3 +/- 5.8% (mean +/- standard error of the mean). Treatment with remacemide hydrochloride significantly ameliorated vasospasm (27.3 +/- 5.4%, p < 0.001). These findings suggest that in this model EAAs may cooperate in the genesis of SAH-induced cerebral vasospasm and that NMDA receptor antagonism with remacemide hydrochloride can partially prevent the SAH-induced vasospasm of a large cerebral artery.

Acetamides↗
PubMed1993

Intracellular calcium dynamics and cerebral injury: modeling various insults in vitro.

The magnitude and time course of intracellular [Ca2+]i alterations were studied after excitatory amino acid challenge (EAA) or chemical energy depletion in mature spinal cultures. While either cytotoxic event led to prompt increases in [Ca2+]i, the pattern of these changes before and after exposure to the toxin was different. EAA [Ca2+]i changes seem primarily dependent on surface membrane alterations from which the cells rapidly recover while energy depletion effects release of [Ca2+]i from intracellular stores and produces a lasting compromise in the ability of these neurons in culture to recover from the initial insult.

Brain Injuries↗
PubMed1993

Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures.

The phospholipid and phospholipid fatty acid compositions of mixed murine spinal cord neuronal cultures are reported. The phospholipid composition was primarily comprised of ethanolamine glycerophospholipids (44.8%) and choline glycerophospholipids (43.5%). Plasmalogens made up 29.1% of the ethanolamine glycerophospholipids (13.0% of the total phospholipids) and 4.5% of the choline glycerophospholipids (1.9% of the total phospholipids). Other phospholipids ranged from 2.9% for sphingomyelin to 1.0% for phosphatidylinositol 4-phosphate. The fatty acid compositions of the ethanolamine glycerophospholipids, choline glycerophospholipids, phosphatidylserine, and phosphatidylinositol were also determined. The choline glycerophospholipids were the most saturated and contained the smallest amount of polyunsaturated fatty acids. The ethanolamine glycerophospholipids were the most unsaturated and contained the highest amount of polyunsaturated fatty acids. The phospholipids contained minimal amounts of 20:3 n-9 (Mead acid) and are not considered polyunsaturated fatty acid deficient. Thus, for the mixed neuronal spinal cord cultures, the phospholipid fatty acid compositions were not polyunsaturated fatty acid deficient and contained a large amount of polyenoic fatty acids of both the n-3 and n-6 series.

Animals↗
PubMed1993

Effect of glutamate and its analogs on diacylglycerol and monoacylglycerol lipase activities of neuron-enriched cultures.

Neuron-enriched cultures from fetal mouse spinal cord contain diacylglycerol and monoacylglycerol lipases. The treatment of neuron-enriched cultures with glutamate or NMDA resulted in a dose- and time-dependent stimulation in diacylglycerol and monoacylglycerol lipase activities. The elevation in the activities of lipases was blocked by the NMDA receptor antagonists, dextrorphan and MK-801, suggesting that lipase stimulation is a receptor-mediated process. The treatment of neuron-enriched cultures with kainate had no effect on diacylglycerol and monoacylglycerol lipase activities. The stimulation of diacylglycerol and monoacylglycerol lipase activities by glutamate and NMDA suggests that these enzymes may play an important role in processes mediated by the NMDA type of the glutamate receptors.

Animals↗
PubMed1993

Cytotoxicity of aluminum silicates in primary neuronal cultures.

To study their cytotoxicity, clays containing aluminum silicates were added to cultures of primary murine spinal cord neurons and differentiated N1E-115 neuroblastoma cells. Bentonite (0.1 mg/ml) and montmorillonite (0.1 mg/ml) rapidly associated with the outer membrane of both N1E-115 and neuronal cells. Erionite (0.1 mg/ml) was randomly distributed throughout the culture. Both bentonite and montmorillonite caused complete cell lysis in the neuronal cultures within 60 min following addition. Erionite had no effect. None of the clays appeared to be cytotoxic to the differentiated N1E-115 cells even though bentonite and montmorillonite were closely associated with the cell membrane. N1E-115 cell lysis did not occur up to 18 h after addition of the clay. Aluminum silicate-containing clays caused a rapid lysis of primary neuronal cells. Differentiated N1E-115 neuroblastoma cells were not susceptible to clay-induced lysis, suggesting that the lytic mechanism is not a general phenomenon that affects all cell types equally.

Aluminum Silicates↗
PubMed1993

Interaction between free radicals and excitatory amino acids in the blood-brain barrier disruption after iron injury in the rat.

Excitatory amino acids and oxygen free radicals have been reported to cooperate in the genesis of brain injury in vivo and in vitro. In this study, we tested the capacity of a noncompetitive N-methyl-D-aspartate receptor antagonist, MK-801, and a 21-aminosteroid, U-74006F, tirilazad mesylate, to block the opening of the blood-brain barrier after subarachnoid injection of FeCl2, which is believed to cause a primarily "pure" free radical insult. Subarachnoid injection of FeCl2 resulted in a significant 10-fold increase in Evans blue extravasation while sham injection or NaCl injection had no effect. Pretreatment with either MK-801 or U-74006F significantly reduced the FeCl2-induced increase in capillary permeability by 43 and 63%, respectively (p < 0.05). Combined treatment with MK-801 and U-74006F resulted in a 65% reduction in vascular leakage that was not significantly greater than pretreatment with either drug alone. These results show that both excitatory amino acids and free radicals can damage the cerebral microvasculature and that an excitatory amino acid antagonist can partially protect the blood-brain barrier after free radical-induced injury.

Amino Acids↗
PubMed1993

The inotropic and beta blocking effects of a chimeric molecule that putatively inhibits both type III phosphodiesterase and beta adrenoceptors in anesthetized dogs.

The hemodynamic and beta adrenergic blocking effects of GI104313, a chimeric molecule containing a phosphodiesterase-inhibiting pyradazinone and a beta blocking phenoxpropanolamine, were examined in barbiturate-anesthetized, vagotomized dogs. The results of these studies were compared to those of indolidan, a known phosphodiesterase inhibitor, and xamoterol, a partial beta adrenoceptor agonist. The compounds were infused at six increasing dose rates in 10-min intervals. Isoproterenol (0.5 microgram/kg) was administered before each dose increment to determine beta adrenoceptor responsiveness. In a separate set of experiments, the hemodynamic effects of GI104313, indolidan and xamoterol were examined in the presence of complete beta blockade with atenolol. GI104313 elicited dose-dependent increases in heart rate, contractility (+dP/dt) and cardiac output and decreases in arterial blood pressure, left ventricular end diastolic pressure and systemic vascular resistance in unpretreated and atenolol-pretreated dogs. However, GI104313 was less potent hemodynamically in atenolol-pretreated animals. This was evidenced by a 4-fold dextral shift in the dose-response relation for several hemodynamic variables. In unpretreated dogs, GI104313 elicited potent dose-dependent blockade of the heart rate, diastolic blood pressure and +dP/dt responses to isoproterenol. Greater than 95% inhibition of isoproterenol response was attained at 1 mumol/kg GI104313 for all observed variables. Indolidan increased contractility and heart rate and decreased diastolic blood pressure in a dose-related fashion. Indolidan did not modify the stimulatory effects of isoproterenol. Atenolol had modest effects on indolidan's hemodynamic effect, only shifting its inotropic effect 2-fold. Xamoterol produced hemodynamic and beta blocking effects similar to GI104313.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Antagonists↗
PubMed1992

Fetal cell grafts into resection and contusion/compression injuries of the rat and cat spinal cord.

This article reviews recent findings concerning the feasibility, basic neurobiology, and potential functional benefits of fetal CNS tissue grafts into acute and chronic lesions of the adult spinal cord. In the rat, neuro-anatomical observations suggest that transplants into resection cavities establish neuritic projections that could functionally reunite separated rostral and caudal segments of the host spinal cord. Furthermore, some complementary electrophysiological evidence has been obtained for synaptic connectivity between host and graft neurons. In these studies, extracellular single-unit activity was evoked in fetal spinal cord (FSC) transplants by stimulating host dorsal roots that had been juxtaposed to donor tissue at the time of transplantation. In other investigations, we examined whether grafts could also establish axonal projections to appropriate areas of gray matter in the chronically injured spinal cord. For this purpose, fetal serotoninergic (5-HT) neurons were injected caudal to complete spinal cord transections that had been made 1-3 months earlier. Immunocytochemistry revealed that these cells projected their axons into gray matter regions normally innervated by bulbospinal 5-HT neurons. To investigate transplantation in a more clinically relevant lesion model, a third group of experiments involved injection of dissociated cell suspensions into acute [less than 24 h postinjury (p.i.)]), subchronic (7-10 days p.i), and chronic (greater than or equal to one month, p.i.) contusion lesions. Such grafts routinely filled areas that otherwise would have been regions of cavitation extending rostral-caudal distances of approximately 7 mm. FSC transplants in such injuries also appeared to influence some aspects of motoneuron excitability and hindlimb locomotion. More recent studies of the cat spinal cord have extended these findings in the rat by showing long-term survival (greater than 2 years) of fetal CNS allografts in recipients with either subtotal transection or compression lesions. Preliminary studies of connectivity have also shown host-graft projection patterns similar to those seen in the rat. Behavioral analyses are currently underway to examine the effects of fetal grafts in cats with chronic postcompression lesions. These observations in the rat and cat are discussed in the general context of basic biological and clinical issues relevant to the long-term objective of promoting functional improvement in the damaged spinal cord.

Animals↗
PubMed1992

In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord.

Magnetic resonance (MR) imaging was evaluated for its possible diagnostic application in determining the survival of fetal central nervous system tissue grafts in the injured spinal cord. Hemisection cavities were made at the T11-L1 level of eight adult female cats. Immediately thereafter, several pieces of tissue, either obtained from the fetal cat brain stem on embryonic Day 37 (E-37), from the fetal neocortex on E-37, or from the fetal spinal cord on E-23, were implanted into the cavities made in seven cats. The eighth cat served as a control for the effect of the lesion only. In another group of four animals, a static-load compression injury was made at the L-2 level. Seven weeks later, the lesion was resected in three cases and fragments of either fetal brain-stem or spinal cord tissue were introduced. A small cyst was observed in a fourth cat in the compression injury group and a suspension of dissociated E-23 brain-stem cells was injected into this region of cavitation without disturbing the surrounding leptomeninges. Five months to 2 years posttransplantation, MR imaging was performed with a 2.0-tesla VIS imaging spectrometer by acquiring multislice spin-echo images (TR 1000 msec, TE 30 msec) in both the transverse and sagittal planes. Collectively, these intermediate-weighted images revealed homogeneous, slightly hyperintense signals at the graft site relative to the neighboring host tissue in seven of the 11 graft recipients. Two of the remaining four cats exhibited signals from the graft site that were approximately isointense with the adjacent host spinal cord, and the final two cats and the lesion-only control presented with very hypointense transplant/resection regions. The hyperintense and isointense images were tentatively interpreted as representing viable graft tissue, whereas the hypointense transplant/resection sites were considered to be indicative of a lack of transplant survival or the absence of tissue in the lesion-only control animal. Postmortem gross inspection of fixed specimens and light microscopy verified the MR findings in the control animal in 10 of the 11 graft recipients by showing either transplants and/or cysts corresponding to the MR images obtained. In one cat in the hemisection group, histological analysis revealed a very small piece of graft tissue that was not detected on the MR images. Therefore, it is suggested that within certain spatial- and contrast-resolution limits, MR imaging can reliably detect the presence of transplanted neural tissue in both the hemisected and compression-injured spinal cord of living animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Neural tissue transplantation and CNS trauma: anatomical and functional repair of the injured spinal cord.

Neural tissue transplantation has become recognized widely as a powerful experimental tool for studying structure-function relationships, development, plasticity, and capacities for regeneration in the adult CNS. In addition, this area of investigation has generated considerable interest in approaches that might be applicable to a variety of catastrophic neurological disorders. In this regard, attention has been given to neural tissue grafting as a potential therapeutic strategy in various forms of neurodegenerative disease. More recently, however, other investigations have begun to focus on the possible application of peripheral and central neural tissue transplants for promoting repair in forms of CNS trauma. This review highlights various neural transplantation approaches that have been explored primarily in the context of injury to the adult CNS, with emphasis on spinal cord injury. An overview is presented of the evolution of this area of research in terms of emerging biological perspectives, technological advances, and experimental modelling. Discussion centers on progress that has been made and a variety of theoretical and practical issues that remain to be resolved.

Animals↗
PubMed1992

Biochemistry and pharmacology of lipid antioxidants in acute brain and spinal cord injury.

Oxygen radical-mediated lipid peroxidation (LP) has been suggested increasingly to be an important factor in posttraumatic neuronal degeneration. Thus, numerous studies have evaluated the neuroprotective efficacy of pharmacological agents with lipid antioxidant activity in models of spinal cord and brain injury. Intensive pretreatment of animals with the endogenous lipid peroxyl radical scavenger vitamin E (i.e., alpha-tocopherol) has been shown to decrease posttraumatic spinal cord ischemia and to enhance chronic neurological recovery. However, the slow CNS tissue uptake of vitamin E requires chronic dosing, making it an impractical agent for treatment of acute neural injury. The glucocorticoid steroid, methyl-prednisolone (MP), has been shown to possess significant antioxidant efficacy and, when administered to animals or humans in antioxidant doses, improves chronic neurological recovery after spinal cord injury. This activity of MP is independent of the steroid's glucocorticoid receptor-mediated actions, as evidenced by the efficacy of the novel antioxidant 21-aminosteroids, which are devoid of glucocorticoid activity but have greater antioxidant efficacy than MP. One of these, tirilazad mesylate (U-74006F), has been shown to be effective in animal models of brain and spinal cord injury and is currently the subject of phase II clinical trials. Recently, compounds that combine the amino functionality of the 21-amino-steroids with the peroxyl radical scavenging chromanol portion of vitamin E (i.e., 2-methylaminochromans) also have shown promise as neuroprotective agents. The consistent benefit afforded by antioxidant compounds further supports the concept that LP is an important therapeutic target for acute pharmacological neuroprotection.

Animals↗