Biomedical subjects

H M Schipper

Publications and source records attributed to H M Schipper.

At least 55 records · Page 3Linked to original sources

PubMed1995

Differential effects of cysteamine on heat shock protein induction and cytoplasmic granulation in astrocytes and glioma cells.

The sulfhydryl agent, cysteamine (CSH), promotes the accumulation of autofluorescent, peroxidase-positive cytoplasmic granules in cultured astroglia akin to those which naturally accumulate in astrocytes of the aging periventricular brain. Both in vitro and in situ, CSH rapidly induces various heat shock proteins (HSP) in astrocytes long before granulation occurs. In the present study, we determined that CSH treatment resulted in an increase in HSP 27, HSP 90 and heme oxygenase (HO-1) at both the protein and mRNA level. We also showed that C6 glioma cells, unlike primary astrocytes, constitutively express HSP 27, HSP 90 and HO-1 at low levels. Moreover, CSH is incapable of eliciting further HSP expression or inducing granulation in the glioma cells. Our results support the hypothesis that the biogenesis of redox-active astrocytic inclusions in CSH-treated glial cultures and in the aging periventricular brain is dependent on an antecedent cellular stress response.

Animals↗

PubMed1995

Redox perturbations in cysteamine-stressed astroglia: implications for inclusion formation and gliosis in the aging brain.

The aminothiol compound, cysteamine (CSH), induces astrocyte hypertrophy (gliosis) and the appearance of autofluorescent, peroxidase-positive cytoplasmic granules in these cells akin to changes that occur spontaneously in astroglia of the aging periventricular brain. Paradoxically, CSH damages astroglial mitochondria (granule precursors) while protecting these cells from subsequent H2O2 and mechanoenzymatic stress. In this study, in vitro CSH administration significantly increased manganese superoxide dismutase (MnSOD) activity in cultured astroglia. Immunoblot and Northern analyses indicated that MnSOD protein and mRNA levels were increased in cultured astrocytes after 3-6 days of CSH treatment. Systemic administration of CSH also significantly augmented MnSOD activity in the intact diencephalon. CSH caused a pronounced (6-fold), but transient, increase in the level of reduced glutathione (GSH) in cultured astrocytes. In contrast, catalase and glutathione reductase (GR) activities were suppressed, whereas copper-zinc superoxide dismutase (CuZnSOD) activity remained unchanged both in cultured astroglia and in the intact diencephalon following CSH treatment. Glutathione peroxidase (GP) activity was increased after 3 and 48 h of CSH treatment and then declined below control levels in cultured astrocytes. CSH inhibited the formation of thiobarbituric acid-reactive products (TBAR) in whole astrocyte monolayers, although it promoted TBAR formation in suspensions of isolated astroglial mitochondria. CSH-related oxidative stress may accelerate aging-related changes in astroglial mitochondria while conferring cytoprotection to these cells by stimulating the upregulation of various heat shock proteins and MnSOD. These cytoprotective responses may facilitate astrocyte survival and the development of reactive gliosis in the face of concomitant neuronal degeneration. CSH-treated astrocytes may serve as a model for the (dys)regulation of neuroglial MnSOD and other antioxidant enzymes in the aging and degenerating nervous system.

Aging↗

PubMed1995

A cellular stress model for the sequestration of redox-active glial iron in the aging and degenerating nervous system.

The mechanisms responsible for the accumulation of redox-active brain iron in normal senescence and in Parkinson's disease remain poorly understood. The aminothiol compound cysteamine (CSH) induces the appearance of autofluorescent, iron-rich cytoplasmic granules in cultured astroglia that are identical to glial inclusions that progressively accumulate in the aging periventricular brain. Both in situ and in culture, these glial inclusions appear to arise in the context of a generalized cellular stress (heat shock) response. Several laboratories have previously concluded that porphyrins and heme ferrous iron are responsible, respectively, for redorange autofluorescence and nonenzymatic peroxidase activity in the glial inclusions. In the present study we found that, contrary to hypothesis, CSH suppresses the incorporation of the heme precursors delta-amino[14C]-levulinic acid and [14C]glycine into astroglial porphyrin and heme in primary culture. Similar results were obtained when the cells were preloaded with radiolabeled heme precursors for 24 h before CSH treatment, suggesting that the latter directly inhibits porphyrin-heme biosynthesis rather than limiting precursor uptake by these cells. We also demonstrated that CSH exposure results in the sequestration of iron-59 by astroglial mitochondria (granule precursors). The results of this study suggest that stress-related trapping of nonheme iron by astroglial mitochondria may be an important mechanism underlying the pathological accumulation of redox-active iron in the basal ganglia of subjects with Parkinson's disease. CSH-treated astrocytes provide a useful model to investigate the role of stress-related dysregulation of neuroglial iron metabolism in the aging and degenerating nervous system.

Aging↗

PubMed1995

Experimental induction of corpora amylacea-like inclusions in rat astroglia.

Corpora amylacea (CA) are glycoproteinaceous inclusions that accumulate in the human central nervous system during normal ageing, and to an even greater extent in Alzheimer's disease and other neurodegenerative disorders. They are particularly prominent in subpial and subependymal regions, and are most commonly located within astrocytes and their processes. We previously demonstrated that human CA share many tinctorial and histochemical properties in common with Gomori-positive cytoplasmic granules which accumulate in periventricular astrocytes of the ageing vertebrate brain and in rat astroglial cultures exposed to the sulphydryl agent, cysteamine (CSH). In the present study, long-term exposure of neonatal rat astrocyte cultures to CSH resulted in the formation of large spherical, PAS-positive cytoplasmic inclusions which are highly reminiscent of, if not identical to, human CA. As in the case of human CA and Gomori-positive astrocyte granules, the CSH-induced CA-like inclusions exhibit non-enzymatic peroxidase activity and consistent immunolabelling with antibodies directed against the mitochondrial protein, sulphite oxidase. Taken together, our findings suggest that progressive mitochondrial damage and macroautophagy play an important role in the biogenesis of CA (and Gomori-positive granules) in astrocytes of the ageing periventricular brain.

Animals↗

PubMed1995

Uptake and subcellular distribution of 51Cr in Gomori-positive astrocytes in primary culture.

An unusual population of astrocytes containing Gomori-positive inclusions occurs in periventricular regions of the brain in all mammalian species. The inclusions are autofluorescent and exhibit non-enzymatic peroxidase activity. Estradiol treatment in vivo and cysteamine treatment in vitro have been shown to increase the number and size of these inclusions. Recent studies indicate that the Gomori inclusions are accumulations of autophagocytized abnormal mitochondria. The mitochondrial changes initiating Gomori inclusion formation begin with a loss of cristae. Energy dispersive X-ray microanalysis also reveals small emission peaks indicative of chromium. The appearance of chromium peaks in the initial stages of mitochondrial transformation suggests that enhanced permeability to chromium could play a causal role in generating Gomori inclusions. In the present study, we have examined the uptake and intracellular distribution of chromium during Gomori inclusion formation in cysteamine-treated cultured astrocytes. 51Cr was added to the media of glial cultures 24 hours prior to the initiation of the formation of Gomori inclusions by the addition of cysteamine. Cultures were fixed and prepared for EM radioautography at 12, 24, and 72 hours following the addition of cysteamine. 51Cr was added to control cells but they were not treated with cysteamine, and, they did not, therefore, develop Gomori inclusions. Cysteamine exposure resulted in a rapid sustained increase in radiolabel over the astrocytes. Much of the label was concentrated over mitochondria. At the late time points, label concentrated progressively over developing Gomori inclusions. These results confirm that the onset of Gomori inclusion formation coincides with increase cellular permeability to chromium and they indicate that uptake of chromium by mitochondria may play an important role in initiating development of these structures.

Animals↗

PubMed1995

Nitric oxide mediated erectile activity is a testosterone dependent event: a rat erection model.

Classically, androgens were thought to be linked to sexual activity in man through their action on increased libido. Recently, the sex hormone dependent nature of nitric oxide synthase (NOS), the enzyme system producing the neurotransmitter of erection (nitric oxide) has been reported. Our study evaluated how changes in testosterone levels alter erectile function. In forty-seven rats the erectile response to cavernous nerve electrostimulation was recorded 1, 5, 10, 20 and 30 d post-bilateral orchiectomy, and compared to controls. Penile tissue was subsequently stained for the presence of NOS, using an NADPH diaphorase technique. Forty eight rats were used in part two. After orchiectomy exogenous testosterone was administered and the erectile function as well as density of NOS positive nerve fibers was assessed. All castrated animals showed a rapid decrease in serum free testosterone levels within 24 h. In contrast, a gradual decrease in intracavernous pressure was recorded with cavernous nerve stimulation, proportional to the time post orchiectomy. NADPH diaphorase staining showed a decreased density of nonadrenergic noncholinergic (NANC) nerve fibers innervating the cavernosal tissue proportional to the time post orchiectomy. With reconstitution of the androgen mileu the erectile response returned to near normal values and recovery of NADPH-positive nerve fibers was observed. Based on presented data we conclude that testosterone or a metabolite plays a direct role in erection acting through an effect on nitric oxide synthase within the corpora cavernosa.

Animals↗

PubMed1994

The 21-aminosteroid antioxidant, U74389F, prevents estradiol-induced depletion of hypothalamic beta-endorphin in adult female rats.

A single intramuscular injection of 2 mg estradiol valerate (EV) results in neuronal degeneration and beta-endorphin depletion in the hypothalamic arcuate nucleus of adult female rats. We have hypothesized that peroxidase-positive astrocytes in this brain region oxidize estrogens and catecholestrogens to semiquinone radicals which mediate oxidative neuronal injury. In the present study, dietary administration of the potent antioxidant 21-aminosteroid, U-74389F, completely blocked EV-induced beta-endorphin depletion in the hypothalami of adult female rats. Neither EV nor 21-aminosteroid treatment had any effect on hypothalamic concentrations of neuropeptide Y and Met-enkephalin, confirming that the estradiol lesion is fairly selective for the beta-endorphin cell population. The present findings support the hypothesis that the toxic effect of estradiol on hypothalamic beta-endorphin neurons is mediated by free radicals.

Animals↗

PubMed1994

A method for analysis of gene expression patterns.

mRNA can be copied into cDNA with the use of reverse transcriptase so that the relative abundance of individual mRNAs is reflected in the cDNA product. With further manipulation a replica of the mRNA expression pattern can be duplicated into a radioactive double-stranded DNA probe. DNA from a series of genes inserted into plasmids can be fixed to a membrane using a slot blot manifold and probed with the RNA-derived DNA probe. The intensity of the hybridization signal for a given gene is a result of its relative abundance in the RNA-derived DNA probe. Quantitation can be achieved through the use of housekeeping genes as baseline monitors. Inclusion of vector sequences can negate any spurious hybridization to vector rather than insert sequences. We have successfully used this method to obtain gene expression patterns for RNA isolated from diverse sources including rodent tissues, various cell lines, and Drosophila and Caenorhabditis elegans samples. Northern blots have verified the results obtained. The pattern of expression of many genes can be determined from as little as 10 micrograms of total RNA, making this method ideally suited for studies in which RNA is rare or in short supply.

Base Sequence↗

PubMed1994

Expression of terminin, a senescence-related cytoplasmic protein, in the aging rat brain.

Terminin is a cytoplasmic protein expressed in irreversibly growth-arrested senescent fibroblast cultures and in terminally differentiated cells of various epithelia. In the present study, terminin was identified by immunohistochemistry in the cytoplasm of neurons and glia of aging rat brain using the monoclonal antibody (Mab) 1.2. Few terminin-positive neurons were observed in 3-month-old brain. At 18 months, terminin immunoreactivity was noted in dentate gyrus granule cells, in hippocampal fibre projections and in neuronal perikarya of deep cerebellar nuclei (but not in cerebellar cortex). In 33-month brain, terminin immunoreactivity in the dentate gyrus was more intense than at 18 months but immunoreactive fibre bundles in the hippocampus were no longer seen. At 33 months, the cerebellar granule cell layer contained terminin-positive horizontal interneurons and received immunoreactive axonal projections not seen in the younger preparations. In addition, ubiquitous low-level terminin expression was noted in neurons of the cerebral cortex, hippocampus and cerebellum of the 33-month-old animals. Thus, in the rat CNS, an increase in terminin appears to be a physiologic marker of neuronal aging. Small numbers of terminin-positive neuroglia were present in gray and white matter of 3-month-old brain and became increasingly more abundant in the 18- and 33-month-old animals. However, even in senescent brains, terminin-positive neuroglia represented a very small fraction of the entire glial pool. Terminin expression in slowly renewing neuroglial populations may identify those cells in which degeneration and death are imminent.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1994

The origin and composition of peroxidase-positive granules in cysteamine-treated astrocytes in culture.

Gomori astrocytes, which are prominent in periventricular regions of the brain, contain inclusions that stain with Gomori dyes, and exhibit an orange-red autofluorescence and a non-enzymatic peroxidase activity. Recently, such astrocytes have been induced in dispersed glial cultures by exposure to cysteamine. Using these cells, we have shown that the peroxidase-positive inclusions (Gomori bodies) are multicompartmental, that iron co-localizes with the peroxidase activity, and that the iron is often segregated in one of the compartments of the body. The goal of the present study was to determine the origin and process of formation of these bodies. The results indicate that cysteamine induces aberrations in mitochondrial structure associated with the acquisition of iron and the associated peroxidase activity. Mitochondria thus transformed appear to initiate an autophagic process in which they, and adjacent structures, are sequestered. The presence of acid phosphatase activity in a number of mature Gomori bodies attests to the participation of lysosomal elements in this process. These results indicate, therefore, that the Gomori body is a complex autophagosome in which the iron-containing compartments, putatively responsible for the peroxidase activity, represent undegraded transformed mitochondria.

Acid Phosphatase↗

PubMed1994

Composition of Gomori-positive inclusions in astrocytes of the hypothalamic arcuate nucleus.

BACKGROUND: Astrocytes within the hypothalamic arcuate nucleus contain Gomori-positive inclusions that exhibit a nonenzymatic peroxidase activity. The source and composition of these Gomori-positive inclusions are currently unknown. Recent evidence, derived from cultured astrocytes, suggests that Gomori-positive inclusions may consist of autophagocytized accumulations of altered mitochondria and that the peroxidase activity is generated by iron or other metals which accumulate in these mitochondria. METHODS: The present study applies electron microscopy, energy dispersive X-ray microanalysis, and immunocytochemistry in conjunction with confocal microscopy to determine the structure and composition of Gomori-positive inclusions in vivo. RESULTS: The results indicate that Gomori-positive inclusions are heterogeneous structures often associated with microtubules and that they contain conspicuous mitochondrial components. Gomori-positive inclusions exhibit X-ray emission peaks for copper and, less often, chromium, either of which could account for the peroxidase activity. CONCLUSIONS: These results support the hypothesis that Gomori-positive inclusions are autophagosomes in which mitochondria are prominent.

Animals↗

PubMed1994

Statin--a novel marker of nonproliferation. Expression in nonneoplastic lymphoid tissues and follicular lymphomas.

Statin is a 57 kDa nuclear protein exclusively found in noncycling cells. Its expression in hematolymphoid cells had not been examined previously. The authors studied statin immunoreactivity in nonneoplastic lymphoid tissues from 26 lymph nodes, 3 tonsils, and 2 spleens. Statin was detected primarily in small lymphoid cells and histiocytes, along with such accessory elements as fibroblasts and endothelial cells. The distribution of positive cells showed an inverse correlation with the proliferative activity of the various lymphoid compartments. Statin labeling was also quantified in 15 follicular lymphomas and compared with that in hyperplastic follicles. Although reactivity in benign germinal centers was less than that in neoplastic follicles, this difference did not prove statistically significant. Statin provides an alternative to proliferation-associated parameters, such as Ki-67 and proliferating cell nuclear antigen, in the analysis of hematolymphoid processes and may be helpful in the dissection of their kinetic heterogeneity.

Cell Cycle Proteins↗

PubMed1993

Stress protein co-localization to autofluorescent astrocytic inclusions in situ and in cysteamine-treated glial cultures.

In the aging brain, a unique subpopulation of limbic and periventricular astrocytes accumulates red autofluorescent, peroxidase-positive cytoplasmic inclusions distinct from lipofuscin. Cysteamine (CSH) exposure rapidly induces identical inclusions in cultured, immature astroglia. CSH induces a cellular stress response prior to astrocyte granulation. To determine whether stress proteins are actual constituents of the autofluorescent granules, 12-week-old rat brain sections and CSH-treated astroglial cultures were immunostained with various anti-stress protein antibodies and evaluated by laser scanning confocal microscopy. We observed intense co-localization of heat shock protein (HSP) 27 and ubiquitin (Ub) to the autofluorescent astrocyte granules in situ and in CSH-treated glial cultures. In both preparations, glucose regulated protein (GRP) 94 consistently exhibited partial co-localization to the granule periphery and adjacent cytoplasm. In contrast, HSP72 co-localization to these inclusions was only occasionally seen and the granules appeared entirely devoid of HSP90 and alpha B-crystallin. Acute exposure of cultured astroglia to CSH induced intense cytoplasmic Ub staining, suggesting that activation of the Ub pathway may be an early event in the biogenesis of these astrocytic granules. Taken together, our results support the notion that the autofluorescent astrocyte inclusions are stress or heat shock granules which progressively accumulate in the aging periventricular brain. Moreover, CSH greatly accelerates the appearance of this senescent astrocyte phenotype in primary culture.

Animals↗

PubMed1993

Isolation of pseudoperoxidase-positive astrocyte granules from intact rat brain and cysteamine-treated neuroglial cultures.

A subpopulation of astrocytes in periventricular regions of aging brain and in cysteamine (CSH)-treated glial cultures contain autofluorescent cytoplasmic granules that exhibit an affinity for Gomori's chrome alum hematoxylin (CAH), and non-enzymatic peroxidase activity. Although shown to be histochemically distinct from lipofuscin, the lack of pure preparations of these glial inclusions has hindered the elucidation of their precise chemical constituents. Using sucrose gradient fractionation and density centrifugation on percoll, we obtained enriched preparations of astrocyte cytoplasmic granules from intact rat brain and CSH-treated astrocyte cultures. The presence and relative purity of these inclusions were confirmed by laser scanning confocal microscopy for red autofluorescent granules, diaminobenzidine histochemistry for non-enzymatic peroxidase activity and chrome alum hematoxylin (Gomori) staining. In the enriched fractions, the smaller granules (0.5-4.0 microns) were spherical and weakly autofluorescent, whereas larger inclusions (5.0-10.0 microns) tended to be intensely autofluorescent and pleomorphic. As in situ, the purified material was argyrophilic and did not stain for lipids. Isolation of these astrocytic inclusions should permit a more thorough characterization of their biochemical contents.

3,3'-Diaminobenzidine↗

PubMed1993

Colocalization of organelle-specific proteins to autofluorescent astrocyte granules by laser scanning confocal microscopy.

Astrocytes in aging periventricular brain regions and in cysteamine-treated neonatal brain cell cultures contain cytoplasmic inclusions that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. In order to delineate the cellular constituents participating in the biogenesis of these astrocytic inclusions, colocalization of FITC-immunolabeled organelles to the red autofluorescent granules was analyzed using a laser scanning confocal imaging system. Areas of true colocalization exhibited yellow fluorescence which persisted in Z-axis image reconstructions. We observed no colocalization of catalase- and PMP70-positive peroxisomes or the Golgi apparatus to the autofluorescent inclusions. The rough endoplasmic reticulum exhibited infrequent dot-like regions of colocalization consistent with previous electron microscopic observations. A minority of early endosomes colocalized to the autofluorescent inclusions in perinuclear cytoplasm. There was extensive colocalization of lgp120-labeled lysosomes to larger autofluorescent granules in close proximity to the nucleus whereas smaller autofluorescent granules often remained unlabeled. A macroautophagic process involving lysosomes and to lesser extent, early endosomes and the rough endoplasmic reticulum, may participate in the biogenesis of autofluorescent astrocytic inclusions in cysteamine-treated glial cultures and in the aging periventricular brain.

Aging↗

PubMed1993

Pathologic effect of estradiol on the hypothalamus.

Estradiol provides physiological signals to the brain throughout life that are indispensable for the development and regulation of reproductive function. In addition to its multiple physiological actions, we have shown that estradiol is also selectively cytotoxic to beta-endorphin neurons in the hypothalamic arcuate nucleus. The mechanism underlying this neurotoxic action appears to involve the conversion of estradiol to catechol estrogen and subsequent oxidation to o-semiquinone free radicals. The estradiol-induced loss of beta-endorphin neurons engenders a compensatory increment in mu opioid binding in the medial preoptic area rendering this region supersensitive to residual beta-endorphin or to other endogenous opioids. The consequent persistent opioid inhibition results in a cascade of neuroendocrine deficits that are ultimately expressed as a chronically attenuated plasma LH pattern to which the ovaries respond by becoming anovulatory and polycystic. This neurotoxic action of estradiol may contribute to a number of reproductive disorders in humans and in animals in which aberrant hypothalamic function is a major component.

Animals↗

PubMed1993

Role of the cellular stress response in the biogenesis of cysteamine-induced astrocytic inclusions in primary culture.

Cysteamine (CSH; 2-mercaptoethylamine) stimulates the accumulation of peroxidase-positive inclusions in cultured astroglia akin to those observed in the aging periventricular brain. Because CSH induces the synthesis of a stress protein (heme oxygenase) in rat liver, we hypothesized that aspects of the cellular stress response may play a role in the biogenesis of CSH-induced astrocyte granules. In the present study, we performed indirect immunofluorescent staining and immunoblotting for various stress proteins in rat neuroglial cultures. Exposure of astrocyte cultures to CSH enhanced immunostaining for heme oxygenase-1 (HO-1) and heat-shock proteins 27, 72, and 90, but not glucose-regulated protein 94, relative to untreated cultures. CSH-pretreated astrocytes exhibited enhanced tolerance to H2O2 toxicity relative to untreated cells, providing physiological evidence of an antecedent stress response in the former. In addition, exposure for 12 days to H2O2, a known inducer of the stress response, elicited astrocyte granulation similar to that observed with CSH. Chronic induction of HO-1 and other stress proteins may participate in the biogenesis of metalloporphyrin-rich inclusions in CSH-treated astroglial cultures and in astrocytes of the aging periventricular brain.

Animals↗

PubMed1993

Quiescent and cycling cell compartments in the senescent and Alzheimer-diseased human brain.

Statin is a 57-kd nuclear protein expressed exclusively in nonproliferating cells. In the present study, immunohistochemical localization of statin in normal, senescent human brain revealed that virtually all neurons, ependymal cells, vascular smooth muscle cells, and endothelial cells are statin-positive and, hence, postmitotic. As we previously demonstrated in rodents, an unexpectedly large fraction of neuroglial cells throughout the aging human brain is statin-negative (range, 41 to 45%), consistent with the substantial retention of neuroglial proliferative capacity well into the senium. In Alzheimer's disease, there is a significant increase in the proportion of statin-negative neuroglia (range, 51 to 58%). In all regions except cerebellum, loss of statin in Alzheimer neuroglia could be accounted for by changes involving the astrocyte subpopulation. These results provide evidence that reactive gliosis in this neurodegenerative disorder encompasses some degree of astrocyte hyperplasia in addition to astrocyte hypertrophy. Maintenance of normal compartments of cycling and quiescent neuroglia in the senescent human brain may serve to define neurologic well-being during the aging process. Conversely, deviations in neuroglial cytokinetics may indicate the presence and extent of intervening neuropathologic processes.

Aged↗