Biomedical subjects

D Maysinger

Publications and source records attributed to D Maysinger.

At least 37 records · Page 2Linked to original sources

PubMed1999

Activation and expression of ERK, JNK, and p38 MAP-kinases in isolated islets of Langerhans: implications for cultured islet survival.

Isolation and purification of islet cells exposes them to ischemic, osmotic and mechanical stresses. The objective of this study was to determine the roles of the MAP-kinases in islets immediately following isolation. During the first 48 h, activity of JNK1 and JNK2 declined markedly. Activity of p38 increased steadily with time in culture while extracellular signal regulated kinase (ERK) activity declined dramatically within 24 h post-isolation. High p38 activation relative to ERK activation immediately following isolation correlated with a decrease in islet survival after 36 h in culture. Absence and/or transiency of ERK signaling in conjunction with sustained activation of p38 pathway could be an important regulator of cell death in islets during and following their isolation by commonly employed procedures.

Adolescent↗

PubMed1999

Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).

Animals↗

PubMed1999

Structural and functional changes resulting from islet isolation lead to islet cell death.

BACKGROUND: Islet isolation exposes the islet to a variety of cellular stresses and disrupts the cell-matrix relationship--events known to be associated with apoptosis. The purpose of this study was to determine whether islet isolation leads invariably to islet cell death and to specify the mechanisms involved. METHODS: Canine islets were isolated using Liberase CI and purified using a centrifuge. Islets were sampled for up to 5 days in culture and analyzed by routine histology, electron microscopy, immunocytochemistry, and reticulin staining for basement membrane. Apoptosis was assessed by cell death enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated decoxyuridine triphosphate nick and labeling (TUNEL) assay. Activation of the prosurvival ERK1/2 and proapoptotic p38 and JNK were determined by immunoblotting. RESULTS: Immediately after isolation, the peri-insular basement membrane was absent, and integrin-alpha 5 expression diminished. DNA fragmentation rose from 2.5 +/- 1.8 (arbitrary units) on the day of isolation to 42.4 +/- 6.7 48 hours later (P < .05), coinciding with the appearance of pyknotic nuclei and apoptotic bodies. The apoptotic index determined by TUNEL assay increased from 5% +/- 1% on the day of isolation to 60% +/- 2% on day 5 (P < .01), and most of the affected cells were beta-cells. Finally, the p38 and JNK activity were elevated relative to ERK1/2. CONCLUSIONS: During isolation, islet cells undergo profound changes in structure and function, resulting in beta-cell apoptosis. These findings suggest that strategies directed to the manipulation of the cell-matrix relationship and the modulation of mitogen-activated protein kinase signal transduction may offer a valuable new approach to improving islet transplant outcome.

Animals↗

PubMed1998

Analysis of c-Fos and glial fibrillary acidic protein (GFAP) expression following topical application of potassium chloride (KCl) to the brain surface.

Application of high K+ concentrations to a limited area of the brain surface is known to trigger spreading depression. We used this model to observe the response of cortical areas, distant to the exposed site, at the cellular level. Immunostaining of glial fibrillary acidic protein (GFAP) and of the proto-oncogene c-Fos was analyzed in brain sections at different times after K+ application. Piriform and parietal cortices, as observed in coronal sections located 3 mm rostrally from the center of the stimulated area and ipsilateral to it, showed a dramatic increase in immunostaining for both markers. However, the time course for such increments was different. c-Fos protein(s) expression was high at 1.5 h and decreased at 24 h after K+ exposure and c-fos mRNA expression correlated with the immunohistochemical results. At these initial times GFAP immunoreactivity was still low but began to rise between 2 and 7 days after treatment in exactly the same areas where c-Fos expression had been up-regulated. No significant effect, for either marker, was evident in the contralateral piriform or parietal cortices. In addition, we studied the effects of the NMDA antagonist MK-801 (4 mg/kg i.p.) on the expression of mRNA for GFAP and c-fos and demonstrated a marked reduction in the upregulation of these genes.

Administration, Topical↗

PubMed1998

From Vanadis to Atropos: vanadium compounds as pharmacological tools in cell death signalling.

Vanadium compounds exert a variety of biological responses, the most notable being their effects as insulin mimetics. More recently, they have been used as pharmacological tools to investigate signalling pathways. Some peroxovanadium compounds act as powerful protein tyrosine phosphatase inhibitors, modulating both the extent and duration of phosphotyrosine signals at the level of the transmembrane growth factor receptors and targets in the cytoplasm and nucleus. A brief history of vanadium compounds, selected chemical properties of vanadium compounds and the ability of peroxovanadium complexes to modulate the activities of protein tyrosine phosphatases and tyrosine kinases are presented in this review by Anne Morinville, Dusica Maysinger and Alan Shaver. From the range of biological activities of these compounds, this review focuses on cytotoxic effects and possible roles of mitogen-activated protein kinases in mediating the effects exerted by vanadium compounds.

Animals↗

PubMed1998

Short communication: transforming growth factor-beta mediates the neurotrophic effect of fibroblast growth factor-2 on midbrain dopaminergic neurons.

Fibroblast growth factor (FGF)-2 is an established neurotrophic factor for dopaminergic (DAergic) neurons in the ventral midbrain. Its survival and differentiation-promoting effects on DAergic neurons in vitro and in vivo are crucially dependent on the presence, numerical expansion and maturation of astroglial cells. We show now that transforming growth factor (TGF)-beta, an established trophic factor for DAergic neurons and product of astroglial cells, mediates the trophic effect of FGF-2 on DAergic neurons cultured from the embryonic rat midbrain floor. Antibodies to TGF-beta that neutralize the isoforms -beta1, -beta2 and -beta3 abolish the trophic effect of FGF-2. FGF-2 increases TGF-beta3 mRNA and amounts of biologically active TGF-beta determined in a mink lung epithelial cell assay in a time-dependent manner. FGF-2 also induces levels of active TGF-beta in neonatal rat astrocytes cultured from midbrain, striatum and cortex. We conclude that TGF-beta is required for mediating the survival promoting effect of FGF-2 on DAergic and, possibly, cortical and striatal neurons grown in the presence of glial cells.

Animals↗

PubMed1997

Role of colony stimulating factor-1 in brain damage caused by ischemia.

A marked effect of colony stimulating factor-1(CSF-1) on microglial response and neuron survival in cerebral cortex ischemic damage was observed. In osteopetrotic op/op mice, which lack systemically functional CSF-1 microglia do not respond to ischemic damage to the cerebral cortex, and the infarcts are considerably larger than in CSF-1 producing mice with similar vascular impairment. Delivery of extraneous CSF-1 to op/op mice alleviates the functional deficiency of the microglia and potentiates neuron survival in ischemic lesion. Delivery of extraneous recombinant CSF-1 to normal CSF-1 producing mice does not increase either the number or degree of activation of microglia, but does further potentiate neuronal survival. We found that neurons in the cerebral cortex have active CSF-1 receptors, and we therefore propose that neuronal rescue in cerebral cortex ischemic damage is linked to activation of the CSF-1 receptor on neurons.

Animals↗

PubMed1997

Drug delivery to the nervous system.

Delivery of drugs to the nervous system remains a challenge despite advances in our understanding of the mechanisms involved in the development of neurodegenerative disorders and the actions of neuroactive agents. Drug accessibility to the central nervous system is limited by the blood-brain barrier; although the peripheral nervous system is more accessible than the central nervous system, problems are still encountered, mainly owing to the poor stability and considerable side effects of many neuroactive compounds when administered systemically. Microencapsulation of neuroactive compounds and living cells producing such substances can overcome some of these shortcomings for delivery to the nervous system.

Animals↗

PubMed1996

Microencapsulated ciliary neurotrophic factor: physical properties and biological activities.

Controlled drug release in the CNS and PNS is still an obstacle to the treatment of neurodegenerative disorders. We have prepared a variety of microspheres containing either ciliary neurotrophic factor (CNTF) or genetically engineered cells able to synthesize and release this cytokine. CNTF is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. However, when given in therapeutically effective doses by systemic injections, it produces numerous adverse side effects. In order to minimize these effects we have microencapsulated it in biopolymers (chitosans, alginates, and copolymers in various proportions to achieve different kinetic properties). Size distribution profiles were determined by an image analysis system and surface characteristics were assessed by electron microscopy. The total content of CNTF as well as the amounts released per day were determined by ELISA and in vitro bioassays. The results from the release kinetics demonstrate that long-term secretion (up to 24 days) of CNTF is achieved by combining chitosan with copolymerized lactic and glycolic acid, whereas microspheres made of alginate provided only relatively short-term release (2-12 days). Neuron survival and neurite outgrowth in cultures of ciliary ganglia were supported by microencapsulated CNTF, indicating biological stability of CNTF. Genetically engineered human kidney cells 293 continued synthesizing CNTF within spheres and the released amounts of CNTF in the culture medium were comparable to the amounts secreted from monolayers (1 ng/ml of supernatant from confluent cultures) or even higher. These studies provide a basis for future testing of CNTF in encapsulated preparations using animal models of neurodegenerative disorders.

Alginates↗

PubMed1996

The hematopoietic cytokine colony stimulating factor 1 is also a growth factor in the CNS: (II). Microencapsulated CSF-1 and LM-10 cells as delivery systems.

The aim of this study was to develop delivery systems for administration of CSF-1 to remedy the systemic deficiency of this cytokine in osteopetrotic op/op mice and to study the microglial response and neuronal survival in op/op mice following cerebral cortex ischemic lesion. Unilateral cerebral cortex ischemic lesions were produced in homozygous op/op mice and either microencapsulated rhCSF-1 or LM-10 fibroblast-like cells producing CSF-1 were administered either locally, at the site and time of the lesioning, or into the peritoneum 2 weeks before the lesion was made. Physical properties (shape, size, integrity) and kinetics of rhCSF-1 release were assessed prior to the experiments in situ. Depending on the characteristics of the biodegradable polymer (e.g., chitosan with different densities or poly-L-lactic-poly-glycolic acid), remarkable differences in survival of encapsulated cells were observed. Cellular integrity following encapsulation and metabolic activity was regularly assessed for a period of 1 month. The best level of viability was achieved with highly viscous chitosan (311). The results from these studies demonstrate that: (1) rhCSF-1 incorporated into biodegradable spheres can be released and retain its biological activity; (2) microencapsulated LM-10 cells which produce CSF-1 can survive and constitutively release CSF-1 in alginate-chitosan spheres for different lengths of time depending on the physical properties of the chitosan used; and (3) CSF-1 is an important growth factor in the central nervous system where it can both strongly alter morphological changes of microglia and enhance survival of neurons in injured brain.

Animals↗

PubMed1996

The survival response of mesencephalic dopaminergic neurons to the neurotrophins BDNF and NT-4 requires priming with serum: comparison with members of the TGF-beta superfamily and characterization of the serum-free culture system.

The neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), are established survival promoting molecules for dopaminergic (DAergic) neurons cultured from the fetal rat midbrain floor. We have cultured and compared the survival of embryonic day (E) 14 mesencephalic cells in fully defined, serum-free medium, with serum-primed cultures (one hour during dissociation). Cultures were characterized using antibodies against neuron-specific enolase (NSE), tyrosine hydroxylase (TH), vimentin, glial fibrillary acidic protein (GFAP), and the antigen A2B5. The absolute absence of serum did not reduce the survival of TH-positive DAergic neurons nor alter the percentages of cells staining for the above markers. Transforming growth factor-beta 3 (TGF-beta 3) and glial cell line-derived neurotrophic factor (GDNF), two members of the TGF-beta superfamily, both promoted the survival of TH-positive cells (TGF-beta 3: 2-fold; GDNF: 1.6-fold) over the 8-day culture period. Survival mediated by TGF-beta 3 and GDNF was independent of whether or not the cells had been initially exposed to serum. In contrast, the survival promoting effects of BDNF and NT-4 were crucially dependent on serum priming. RT-PCR for the full-length trkB high affinity neurotrophin receptor revealed its presence in both culture systems. We conclude that priming with serum is important to make DAergic neurons fully responsive to BDNF and NT-4. Underlying mechanisms might be sought at the level or distal of trkB receptor expression, without excluding the possiblity that serum elicits production of growth factors that synergistically act with neurotrophins in these cultures.

Animals↗

PubMed1996

Colony stimulating factor-1 potentiates neuronal survival in cerebral cortex ischemic lesion.

The effect of the cytokine, colony stimulating factor-1 (CSF-1), on neuronal survival in cerebral cortex ischemic lesion was determined. Ischemic lesions were made in C3H/HeJ mice by disrupting blood vessels that penetrate the cerebral cortex from the pial-vascular plexus. Recombinant human colony stimulating factor 1 (rhCSF-1) was delivered in chitosan microcapsules that were either implanted intraperitoneally 2 weeks before surgery or at the site of the lesion at the time of surgery. Neuronal survival was twofold greater and the size of the infarct was considerably smaller in animals that received rhCSF-1-containing microcapsules. There was no significant difference whether the microcapsules were implanted intraperitoneally or at the site of the lesion. We found that CSF-1 receptor (c-fms) was upregulated in neurons at the site of the lesion and we propose that neuron rescue in ischemic damage is potentiated by CSF-1 signaling through CSF-1 receptor in the neurons.

Animals↗

PubMed1995

Developmental deficiency of the cholinergic system in congenitally hyperammonemic spf mice: effect of acetyl-L-carnitine.

The sparse-fur (spf) mutant mouse has an X-linked deficiency of hepatic ornithine transcarbamylase (OTC) and develops hyperammonemia in the postnatal period similar to that seen in human patients. We studied the effect of congenital hyperammonemia on the development of cerebral cholinergic parameters such as choline acetyltransferase (ChAT), acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) in spf mice. The serum ammonia levels of spf mutant mice were significantly elevated after weaning compared with control animals. ChAT activity levels started decreasing in mutant spf mice from the age of 30 days (i.e., immediately after weaning); it reached significantly lower levels in the adult animals. HACU was consistently lower (P < .01) in spf/Y mice compared with controls up to the adult stage. However, there were no marked changes in the activity of AChE between control and hyperammonemic spf mice. The levels of beta-NGF, which is essential for cholinergic differentiation and function, were significantly lower in different brain regions of adult mutant mice compared with normal controls. A treatment of spf/spf breeding females with acetyl-L-carnitine, at a dose of 1.5 mM in drinking water, starting from day 1 of conception, resulted in a significant restoration of ChAT activity levels in some brain regions of the spf/Y offspring. The beta-NGF levels were also significantly elevated after supplementation with ALCAR in mutant mice compared with untreated mutant mice. These data are suggestive of a neurotrophic property of ALCAR during cholinergic deficiency caused by congenital hyperammonemia.

Acetylcarnitine↗

PubMed1994

In vitro effects of brain derived neurotrophic factor released from microspheres.

The principal objective of these studies was to microencapsulate brain derived neurotrophic factor (BDNF) and to test its biological effects in vitro. Two types of microspheres with BDNF were prepared using biodegradable polymers: chitosan and poly-(L-lactic acid):poly-(L-glycolic acid). Depending on the procedure, sizes ranged from 1 micron to 1 mm as determined by an image analysis system. The biological assays confirmed that the released BDNF remains biologically active in vitro. Microencapsulation of BDNF and other trophic factors may represent a viable alternative to currently employed delivery systems in vivo and holds potential for eventual therapy of neurodegenerative disorders, in particular for peripheral neuropathies and CNS disorders where supplementation of neurotrophins retards or even prevents neural degeneration.

Animals↗

PubMed1994

Neocortical infarction in subhuman primates leads to restricted morphological damage of the cholinergic neurons in the nucleus basalis of Meynert.

The aim of the present study was to investigate the long-term effect of cortical infarction on the subhuman primate (Cercopithecus aethiops) basal forebrain. The lesion, carried out by cauterizing the pial blood vessels supplying the left fronto-parieto-temporal neocortex, induced retrograde degenerative processes within the ipsilateral nucleus basalis of Meynert. The morphometrical analysis revealed that significant shrinkage of cholinergic neurons and loss of neuritic processes were localized within the intermediate regions of the nucleus basalis. The average cross-sectional areas of choline acetyltransferase-immunoreactive neurons in the intermedio-ventral (Ch4iv) and intermedio-dorsal (Ch4id) nucleus basalis were decreased to 62.5 +/- 9.5 and 58.0 +/- 8.6%, respectively, of the sham-operated values. Although an apparent loss of Nissl-stained magnocellular neurons in Ch4iv and Ch4id was found by applying a quantitative analysis based on a perikaryal-size criterion, data obtained by the quantification of immunostained material failed to reveal any significant decrease of cholinergic cell density. Results are discussed in view of future application of this ischemic model to study processes of retrograde degeneration following cortical target removal and to assess potential neurotrophic and neuroprotective properties of pharmacologic agents.

Animals↗

PubMed1994

Preparation, characterization and release of microencapsulated bromodeoxyuridine.

Biodegradable and biocompatible microspheres with bromodeoxyuridine (BrUrd) have been prepared, characterized and tested in vitro and in vivo. Scanning electron microscopy and image analysis revealed regular spherical shapes and an average size +/- SD of 2.47 +/- 0.59 microns. Total content of BrUrd as determined by HPLC was within the range of 0.2-1.5%. Kinetic analyses of two different preparations showed similar release half-times (approx. 12 hrs), kinetic constants -0.0556, -0.0564, and -0.0557, -0.0597 [h-1], and correlation coefficients of 0.998, and 0.999 when fitted to the first order or biphasic first order kinetics, respectively. Preliminary data from immunocytochemical studies revealed efficient incorporation of BrUrd delivered from these microcapsules into nuclei of proliferating cells surrounding brain lesions in rats.

Animals↗

PubMed1994

Encapsulated genetically engineered fibroblasts: release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions.

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable "minifactories" continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving "empty" alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs non-lesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased (P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

Alginates↗

PubMed1994

Chemiluminometric determination of choline-related substances in pharmaceutical preparations by dot-blot.

A simple and reliable method of assaying succinylcholine chloride, oxtriphylline (choline theophyllinate) and acetycholine chloride in pharmaceutical preparations, based on conversion to choline, is provided by combination of chemiluminometry of choline and dot-blot technique. Recoveries of 102 and 97% for choline chloride in Quelicin injection and Choledyl 200 tablets matrices, respectively, and of 99% for acetylcholine chloride in Miochol solution matrix could be achieved using simple choline chloride standards in phosphate buffer pH 8.6. Accordingly, matrix-matched standards were found redundant. Favourable results obtained in preparations-matched media including limits of detection of 37-39 pmol microliters-1 of choline chloride, together with accuracies of 0-2% and RSDs ranging from 4 to 7% are the further evidence of the suitability of the method.

Acetylcholine↗