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Biomedical subjects

I R Brown

Publications and source records attributed to I R Brown.

At least 37 records · Page 2Linked to original sources
PubMed1997

Tissue-specific differences in heat shock protein hsc70 and hsp70 in the control and hyperthermic rabbit.

The ability to resolve protein members of the hsp70 multigene family by two-dimensional Western blotting permitted the characterization of antibodies which were specific in discriminating constitutively expressed hsc70 isoforms from stress-inducible hsp70 isoforms. This antibody characterization demonstrated that basal levels of hsp70 isoforms were present in the cerebellum of the control rabbit and that these were elevated following hyperthermia, whereas levels of hsc70 were similar in control and hyperthermic tissue. Multiple isoforms of hsp70 were detected but tissue-specific differences were not apparent in various organs of the rabbit. However, species differences were observed as fewer hsp70 isoforms were noted in rat and mouse. In the control rabbit, higher levels of hsc70 protein were present in neural tissues compared to non-neural tissues. Following physiologically relevant hyperthermia, induction of hsp70 was greatest in non-neural tissues such as liver, heart, muscle, spleen, and kidney compared to regions of the nervous system. These studies suggest that the amount of preexisting constitutive hsc70 protein may influence the level of induction of hsp70 in the stress response. Given this observation, caution is required in the employment of hsp70 induction as an index of cellular stress since endogenous levels of hsc70, and perhaps hsp70, may modulate the level of induction.

Animals↗
PubMed1997

Differential induction of heat shock mRNA in oligodendrocytes, microglia, and astrocytes following hyperthermia.

A time course analysis of hsp70 mRNA induction in response to a physiologically relevant increase in body temperature of 2.6 degrees C was performed in the rabbit forebrain. A protocol that combined in situ hybridization and cytochemistry on the same tissue section was employed to identify reactive glial cell types. Cytochemical markers for astrocytes, microglia, and oligodendrocytes were utilized in combination with a DIG-labelled hsp70 riboprobe, which permitted mRNA localization at high resolution. Four glial cell body-enriched regions of the rabbit forebrain were examined, namely, cortical layer 1, hippocampal fissure, corpus callosum, and fimbria. Maximal hsp70 mRNA induction was observed in 2 and 3 h hyperthermic animals. The colocalization analysis demonstrated that hsp70 mRNA was induced in oligodendrocytes and microglia, but not in forebrain GFAP positive astrocytes. In addition, cell counts were performed which showed that almost all oligodendrocytes induced hsp70 mRNA while a subpopulation of microglial cells responded. These data are consistent with the notion that oligodendrocytes, microglia, and astrocytes exhibit distinct thresholds for activation of the heat shock response following a physiologically relevant increase in body temperature.

Animals↗
PubMed1997

Developmental analysis of factors binding to the mouse 68-kDa neurofilament promoter.

Whole tissue extracts prepared from mouse brain regions at various postnatal ages were characterized for binding of factors to the DNase I hypersensitive site (HSSI) which is located closest to the transcription start site of the 68-kDa mouse neurofilament gene (NF-L). Gel mobility shift assays detected changes in factor binding during postnatal development of the neocortex. Competition experiments suggested that one of the complexes resulted from factor binding to a 9 bp sequence found in both the light and medium neurofilament promoter regions (NF-L/M). Gel mobility shifts performed with an oligonucleotide probe containing the NF-L/M sequence detected two brain-specific DNA-protein complexes, and a third complex in both brain and liver. During cerebellar and neocortical development, one of the NF-L/M complexes was most intense at postnatal day 10 when transcription of the NF-L gene is upregulated.

Aging↗
PubMed1997

Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola.

The active oxygen species hydrogen peroxide (H2O2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2O2 was typically present within the secondary thickened walls of xylem vessels. Inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls adjacent to attached bacteria. Quantitative analysis indicated a prolonged burst of H2O2 occurring between 5 to 8 hr after inoculation in cells undergoing the HR during this example of non-host resistance. Cell wall alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not associated with intense staining for H2O2, unless the responding cell was undergoing the HR. Catalase treatment to decompose H2O2 almost entirely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibitor) did not affect the pattern of distribution of H2O2 detected. H2O2 production was reduced more by the inhibition of plant peroxidases (with potassium cyanide and sodium azide) than by inhibition of neutrophil-like NADPH oxidase (with diphenylene iodonium chloride). Results suggest that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an excess of H2O2 in the early stages of the plant-bacterium interaction was only produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as the cause of localized membrane damage at sites of bacterial attachment.

Catalase↗
PubMed1996

Intracellular localization of heat shock mRNAs (hsc70 and hsp70) to neural cell bodies and processes in the control and hyperthermic rabbit brain.

Heat shock proteins are essential cellular proteins that may play important roles in cellular repair and/or protection. This report focuses on the expression of two members of the hsp70 multigene family, namely, constitutive hsc70 mRNA and stress-inducible hsp70 mRNA in the control and hyperthermic rabbit brain. The intracellular localization of these heat shock mRNAs was examined using high-resolution nonradioactive in situ hybridization. The distribution of hsc70 mRNA and hsp70 mRNA was examined in (1) neuronal cell bodies and their dendritic processes and (2) oligodendrocytes and their cellular processes. In control animals, hsc70 mRNA was detected in the apical dendritic processes and cell bodies of cortical layer II and V neurons, CA3 and CA4 neurons, deep cerebellar neurons, and brainstem neurons. A time course analysis of hsc70 mRNA, after a physiologically relevant increase in body temperature of 2.6 degrees C, revealed more distal transport of this constitutive message into dendrites of these neuronal populations. In the same neuronal populations, basal levels of hsp70 mRNA were observed in the cell body; however, this mRNA was not detected in dendritic processes in control or hyperthermic animals. After hyperthermia, hsp70 mRNA was strongly induced in oligodendrocytes and transported to the processes of these glial cells. The localization of heat shock messages in the processes of these neural cell types could provide a mechanism for local control of synthesis of heat shock proteins in cellular compartments that are remote from the cell body.

Animals↗
PubMed1996

The neuronal stress response: nuclear translocation of heat shock proteins as an indicator of hyperthermic stress.

Two characteristic features of the heat shock response, (i) induction of hsp70 protein and (ii) nuclear translocation of constitutive hsc70 and stress-inducible hsp70 protein, were utilized as markers of cellular stress in the rabbit brain. Following a physiologically relevant increase in body temperature of 2.7 +/- .3 degrees C, nonneuronal cell types, such as ependymal cells and oligodendrocytes, undergo a stress response as assayed by the above criteria. In contrast, several neuronal cell populations required an increased degree of hyperthermic stress (3.4 +/- .2 degrees C) before exhibiting nuclear translocation of constitutive hsc70 protein. Induction of hsp70 protein was not observed in these neuronal cells at either temperature. The present results suggest that certain neurons in the rabbit brain are buffered against induction of the heat shock response, perhaps due to their high constitutive levels of hsc70 protein.

Animals↗
PubMed1996

SC1, a brain extracellular matrix glycoprotein related to SPARC and follistatin, is expressed by rat cerebellar astrocytes following injury and during development.

In the nervous system, extracellular matrix components are believed to influence cell shape, proliferation and migration during development and following injury. SC1 is a secreted glycoprotein expressed during neural development and in the adult brain. The molecule shows partial sequence homology to the anti-adhesive extracellular matrix molecule SPARC/osteonectin and to follistatin. We have made a surgical lesion in the adult rat cerebellum and examined changes in SC1 expression at 1 to 14 days after injury. Dual in situ hybridization/immunohistochemistry demonstrated that SC1 mRNA was induced in astrocytes surrounding the wound, reaching maximal levels at 10 days post-lesion. Immunohistochemistry revealed changes in the deposition of SC1 protein in radial fibres of Bergmann glia. SC1 protein was also detected at the border of the lesion, suggesting an association with the glial scar. Double immunohistochemistry with the astrocytic marker GFAP demonstrated that astrocytes also express SC1 during postnatal development.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

In vivo activation of neural heat shock transcription factor HSF1 by a physiologically relevant increase in body temperature.

Molecular mechanisms which underlie the heat shock response have commonly been analyzed using tissue culture systems, with less investigation of the intact mammal. In tissue culture, a temperature elevation of 5 degrees C is required to activate mammalian heat shock transcription factor 1 (HSF1) to the DNA-binding form. We demonstrate that a physiologically relevant increase in body temperature of 2.5 +/- 0.2 degrees C, similar to that attained during fever reactions, is sufficient to activate HSF1 in the rabbit nervous system. Maximal HSF activation, as measured by gel mobility shift assay, was attained at 1 hr with the cerebellum showing the strongest signal. Supershift experiments with antibodies specific to HSF1 and HSF2 demonstrated that the signal reflected activation of HSF1. Western blot analysis showed that cerebellum exhibited high levels of HSF1 protein.

Animals↗
PubMed1996

SC1, a SPARC-related glycoprotein, exhibits features of an ECM component in the developing and adult brain.

Although extracellular matrix (ECM) components have been shown to play important roles in the development of the CNS, expression generally decreases in the adult brain. This study examines the expression of the SPARC-related glycoprotein SC1 in the rat brain during postnatal development and in the adult. In situ hybridization analysis indicates that expression of SC1 mRNA increases in a caudal to rostral manner as postnatal neural development proceeds and is found at near maximal levels in the adult brain. SC1 mRNA is expressed in glial-enriched areas of the brain at postnatal day 1 (P1) and P5. Between P10 and P20, SC1 mRNA increases in neuron-enriched regions of the hippocampus, dentate gyrus, and cerebral cortex. Immunohistochemistry in the adult shows that SC1 protein is localized to neurons in these regions and to scattered glial cells. Subcellular fractionation demonstrates that the SC1 116/120 kDa doublet is associated with synaptosomes. SC1 is present in the aqueous phase following extraction of membranes with TX-114, suggesting that it is not a transmembrane protein, a property consistent with other adult brain ECM components. Furthermore in cerebellar granule cells grown in culture, high levels of the 120 kDa component are secreted into the media. These results are consistent with the hypothesis that SC1 is an ECM glycoprotein expressed in both the developing and adult brain.

Animals↗
PubMed1996

CaM I mRNA is localized to apical dendrites during postnatal development of neurons in the rat brain.

In the rat, a single calmodulin (CaM) protein is encoded by three separate genes which produce five different transcripts. The significance of the multiple CaM genes is not known; however, individual CaM transcripts could be targeted to specific intracellular sites. In this report, the cellular distribution of CaM I mRNAs was analyzed in the postnatal rat brain. The 4.0-kb CaM I transcript was present in neuronal cell bodies and also localized to apical dendritic processes. In cerebral cortical neurons, the 4.0-kb CaM I mRNA was detected in apical dendrites at postnatal day (PD) 5 to 15. In hippocampal neurons, this CaM message was present in dendritic processes from PD S to 20, whereas in Purkinje neurons it was detected in dendrites at PD 15 and 20. The presence of the 4.0-kb CaM I mRNA in dendrites of the rat brain supports the notion of targeting transcripts derived from the CaM multigene family to discrete intracellular destinations.

Aging↗
PubMed1996

Alteration of CaM I mRNA expression in the developing rat superior colliculus following chronic treatment with an NMDA receptor antagonist.

The spatial distribution of CaM I mRNA was investigated in the developing superior colliculus of rats that were chronically treated with the NMDA receptor antagonist AP-5. In control animals, CaM I message was localized in a continuous band of cells that extended across the entire rostro caudal axis of the superficial superior colliculus. Chronic AP-5 treatment resulted in a specific reduction of CaM I message in the caudal colliculus at postnatal day 10. Since normal NMDA receptor function has been implicated in activity dependent synaptic plasticity in the superior colliculus, these results suggest that the regulation of calmodulin may be part of a cascade of events that mediate this plasticity following NMDA receptor activation.

2-Amino-5-phosphonovalerate↗
PubMed1996

Expression of mRNA species encoding heat shock protein 90 (hsp90) in control and hyperthermic rabbit brain.

Northern blot and in situ hybridization were employed to investigate regional and cell type differences in the expression of hsp90 mRNA species in control and hyperthermic rabbit brain. Riboprobes specific to hsp90 alpha and beta mRNA species were utilized in time-course Northern blot studies on cerebral hemispheres and the cerebellum. Following hyperthermia, levels of hsp90 alpha and beta mRNA were elevated in both brain regions; however, the magnitude of induction was more robust in the cerebellum than in cerebral hemispheres. The pattern of expression of hsp90 genes in rabbit brain was analyzed by in situ hybridization. These studies revealed that hsp90 genes are preferentially expressed in neuronal cell populations in the unstressed mammalian brain. The distribution of hsp90 alpha and beta mRNA was similar, though the signal for the latter was stronger. Following hyperthermia, changes were not detected in the pattern of hsp90 beta mRNA expression in the hippocampus. In the cerebellum, a rapid induction of hsp90 beta mRNA was apparent in the neuron-enriched granule cell layer, followed by a delayed accumulation in Purkinje neurons. Unlike hsp70, induction of hsp90 was not detected in glial cells of hyperthermic rabbit brain. The localization of hsp90 to neurons suggests that this heat shock protein plays an important role in neuronal function.

Animals↗
PubMed1996

Characterization of DNase I hypersensitive sites in the mouse 68-kDa neurofilament gene.

Four brain-specific DNase I hypersensitive sites (HSS) have previously been identified flanking the mouse 68-kDa neurofilament gene within a 1.7 kb upstream sequence which confers neuronal specificity of expression of this gene in transgenic mice. Previously several DNA-binding factors were detected at the HSS closest to the transcription start site (HSS1). However, no major brain-specific factors were identified, suggesting a possible role for the three remaining HSS in conferring tissue-specificity to the NF-L gene. Sequence analysis of the NF-L promoter region demonstrated the presence of an extensive CT repeat and several potential binding sites which are also found in other neurofilament promoters. Gel mobility shift assays revealed a similar but not identical banding pattern with brain and liver nuclear extracts at HSS2, and HSS3, however the banding pattern for HSS4 was predominantly brain-specific. DNase I footprinting revealed several factors binding to the upstream HSS regions in brain and liver nuclear extracts. These include a CCAAT box at HSS2, a novel brain-specific footprint near an adenovirus promoter element E2aE-C beta and a single liver-specific footprint associated with an POU/octamer binding site at HSS4. The presence of brain-specific gel shift bands and tissue-specific footprints associated with HSS4, suggest that this region may play an important role in the regulation of the NF-L gene.

Animals↗
PubMed1995

Localization of constitutive and hyperthermia-inducible heat shock mRNAs (hsc70 and hsp70) in the rabbit cerebellum and brainstem by non-radioactive in situ hybridization.

Neural expression of constitutive hsc70 mRNA and hyperthermia-inducible hsp70 mRNA is examined using radioactive and non-radioactive in situ hybridization procedures. A strong induction of hsp70 mRNA was noted in cell populations in cerebellar layers and in the brainstem which demonstrated expression of mRNA encoding proteolipid protein, an oligodendrocyte marker. The non-radioactive in situ hybridization procedure using digoxigenin (DIG)-UTP-labeled riboprobes permitted improved signal localization, and stress-inducible hsp70 mRNA was detected at the cytoplasmic cap areas of individual oligodendrocytes. Cell types which express constitutive members of the hsc/hsp70 multigene family were also identified. Neurons in the brainstem and in the deep white matter and molecular layer of the cerebellum showed expression of hsc70 mRNA while signal was not detected in adjacent glial cells. A neuron-specific enolase riboprobe aided in the identification of neuronal cell types. The non-radioactive DIG riboprobe revealed that hsc70 mRNA was highly localized to the cytoplasm of individual neurons. High constitutive levels of hsc70 in certain neurons may dampen hsp70 induction after hyperthermia in these cell populations.

Animals↗
PubMed1995

SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain.

Although extracellular matrix (ECM) glycoproteins play important roles in neural development, their levels are generally believed to decrease in the adult brain. Immunohistochemical analysis indicates that the anti-adhesive ECM glycoprotein SPARC/osteonectin, which contains a follistatin 'module,' is expressed in the adult rabbit nervous system. In the cerebellum, SPARC is present in Bergmann glia, with a strong signal along their radial fibres. SPARC, while enriched in membrane fractions, is not a transmembrane protein. In the hippocampus, colocalization of SPARC is observed in cells which express the astrocytic marker GFAP. The expression of SPARC by a subset of astrocytes, particularly in synaptic enriched areas, suggests a continuing role for the ECM in the adult brain.

Animals↗
PubMed1995

Developmental expression of calmodulin mRNA and protein in regions of the postnatal rat brain.

The expression of calmodulin (CaM) protein and mRNA was analyzed in specific regions of the rat brain during postnatal development. CaM levels in the adult brain were more abundant in the cerebral hemispheres and thalamus compared to brain stem and superior plus inferior colliculus. All brain regions contained higher CaM protein and mRNA levels than in non-neural tissues such as the kidney. During postnatal development of the brain, maximal levels of CaM protein and CaM I mRNAs were attained at day 10 or 15. Protein levels declined thereafter in the adult in all regions except the thalamus. With respect to products of the rat CaM I gene, the 4.0 kb neural transcript demonstrated a pronounced increase during postnatal development, whereas the 1.8 kb message showed little change.

Animals↗