Glycoproteins of the post-synaptic density: biochemical and immunochemical characterization of PSD-GP180.
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Animals↗
Biomedical subjects
I R Brown
Publications and source records attributed to I R Brown.
Molecular cloning of neuronal calmodulin mRNA species.
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Animals↗
Cysteamine in combination with N-acetylcysteine prevents acetaminophen-induced hepatotoxicity.
N-Acetylcysteine (NAC) is protective against acetaminophen-induced hepatotoxicity primarily by providing precursor for the glutathione synthetase pathway, while cysteamine has been demonstrated to alter the cytochrome P-450 dependent formation of toxic acetaminophen metabolite. Mice administered acetaminophen (500 mg/kg) had elevations of serum alanine aminotransferase (ALT) to 273.0 +/- 37.5 and 555.8 +/- 193.4 U/mL at 12 and 24 h, respectively, after injection. Administration of cysteamine (100 mg/kg) or NAC (500 mg/kg) significantly reduced serum ALT activity (p less than 0.001). Reducing the dose of NAC or cysteamine by 50% greatly reduced their hepatoprotective effect while the co-administration of the reduced doses of NAC (250 mg/kg) and cysteamine (50 mg/kg) following acetaminophen overdose prevented elevation of serum ALT activity (39.2 +/- 1.17 and 32.5 +/- 5.63 U/mL at 12 and 24 h post-injection, p less than 0.001) and preserved normal mouse hepatic histology. Neither NAC (500 mg/kg), cysteamine (100 mg/kg), or the lower doses in combination of both agents were found to alter the half-life or peak levels of acetaminophen. Liver microsomal aryl hydrocarbon hydroxylase activity measured 24 h after drug administration was not significantly different between treatment groups and controls receiving only saline. These results indicate a possible role for the concomitant use of NAC and cysteamine in the prevention of hepatic necrosis following toxic doses of acetaminophen. Neither decrease in plasma acetaminophen levels nor depression of cytochrome P-450 enzyme activity appears to be the mechanism of protection when these doses of NAC, cysteamine, or both drugs together are administered with a toxic dose of acetaminophen in mice.
Acetaminophen↗
Cell-specific expression of high levels of human S100 beta in transgenic mouse brain is dependent on gene dosage.
The beta-subunit of S100 protein (S100 beta) is highly conserved in the mammalian brain. The gene coding for human S100 beta has been mapped to chromosome 21. In order to study the consequences of overexpression of the S100 beta gene, transgenic mice were generated by microinjection of a 17.3 kilobase human genomic fragment containing the three exons and the transcription control elements of the human S100 beta gene. Mice from four transgenic lines carried approximately 10-100 transgene copies. Northern blotting demonstrated a tissue-specific and gene dose-dependent expression of human S100 beta mRNA in mouse brain. Increased expression of S100 beta mRNA was correlated with an increased production of S100 beta protein. Examination of brain sections by in situ hybridization and immunocytochemistry indicated that S100 beta was localized globally to astrocytes, as well as to discrete neurons in the mesencephalic and motor trigeminal, facial, and lemniscus nuclei in both normal and transgenic mice. In peripheral tissues, human S100 beta was expressed at 10-50-fold lower levels than in brain. The strict gene dosage dependence and cell specificity of transgene expression suggest the presence of a locus control region (LCR) in the human S100 beta gene. The mice tolerated 10-100-fold higher than normal levels of S100 beta gene expression in brain without any gross physical or behavioral abnormalities. The high-level expression and cell specificity of the S100 beta promoter/LCR suggest that it may provide a valuable tool to direct the expression of other transgenic products to specific cell types in the CNS.
Animals↗
Interleukin 1 induces the expression of a heat-shock gene in chondrocytes.
The presence of T cells and antibodies reactive with heat-shock proteins (hsps) in the joints of patients with rheumatoid arthritis may indicate a role of hsps in this disease. In the present study we examined whether increased temperature and interleukin 1 (IL 1), both of which are elevated in arthritic joints, induced the expression of two hsp70 genes in bovine chondrocyte cultures. We found that heat shock resulted in increased expression of constitutive and inducible hsp70 mRNA species. IL 1 and phorbol 12-myristate 13-acetate (PMA) also induced an increase in the constitutive hsp70 mRNA species, but without affecting the expression of the inducible hsp70 gene. The increase induced by IL 1 was observed only after 3 h, whereas increases induced by PMA were observed within 1 h. For all treatments, the hsp70 mRNA decreased by 24 h. Heat treatment of chondrocytes did not affect levels of collagenase and caseinase activity in the medium, nor did it alter proteoglycan synthesis by these cells.
Animals↗
Expression of heat shock genes (hsp70) in the mammalian nervous system.
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Animals↗
Heterogeneity in expression of S100 beta mRNA in human melanoma and pleomorphic adenoma demonstrated by in situ hybridization.
S100 protein is a widely used immunohistochemical marker for identification of a number of tumors including malignant melanoma and pleomorphic adenoma of the salivary gland. To extend the detection techniques for S100 protein to the level of its mRNA, sections of malignant melanoma and pleomorphic adenoma were hybridized in situ with a 35S-labeled anti-sense RNA probe complementary to the mRNA for the beta subunit of human S100 protein. Both tumors were labeled with the anti-sense RNA probe but not with a sense RNA probe. In addition, sections of normal and tumor tissues which were known not to express S100 protein on the basis of immunohistochemical studies were not labeled with the anti-sense RNA probe. These results established the specificity of the in situ hybridization technique for the detection of S100 protein mRNA. Although most of the tumor cells in both malignant melanoma and pleomorphic adenoma were labeled with the anti-sense RNA probe, unlabeled tumor cells were also present in their vicinity, suggesting there was a heterogeneity among the cells in both tumor types with respect to S100 protein mRNA expression.
Adenoma, Pleomorphic↗
Expression of heat shock genes (hsp70) in the mammalian brain: distinguishing constitutively expressed and hyperthermia-inducible mRNA species.
The mammalian genome contains both constitutively expressed and heat-shock-inducible members of the hsp70 gene family. Riboprobes derived from members of these two classes of heat shock genes were utilized in Northern blot studies to analyze brain mRNA isolated from control rabbits and rats and from animals subjected to hyperthermic treatment. A riboprobe derived from a constitutively expressed hsp70 gene detected a 2.5 kilobase (kb) mRNA in brain tissue from control rabbits and a 2.3 kb mRNA species in control rat brain. These brain mRNAs showed little change in abundance in animals which were subjected to hyperthermic treatment. A riboprobe derived from a heat-shock-inducible hsp70 gene detected an abundant 2.7 kb brain transcript in hyperthermic rabbits which was not apparent in control animals. A time course study revealed that the induction of this mRNA species was transient and paralleled the rise and fall in body temperature. Peak induction was observed at 1 hr. The level of this message had greatly decreased by 5 hr and only trace levels were present at 10 and 24 hr. In the rat brain the induced hsp70 mRNA species was slightly larger than that observed in rabbit (2.9 kb vs. 2.7 kb). The riboprobe which detected the hyperthermia-inducible mRNA species was highly specific and did not cross react to the constitutively expressed mRNA species under the conditions employed in the Northern blot studies.
Animals↗
Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization.
Glial fibrillary acidic protein (GFAP) accumulates in astrocytes during development. We have characterized the increase in GFAP mRNA during development of the rat brain by using Northern blotting and in situ hybridization histochemistry and have found a caudal to rostral gradient of expression, consistent with overall brain maturation. GFAP mRNA was first observed at embryonic day 16 (E16) in the glial limitans of the ventral hindbrain. During brain development message levels increased rostrally and by postnatal day 5 (P5) the entire glial limitans showed a positive signal which persisted into adulthood. GFAP mRNA was also found to accumulate in a caudal to rostral direction within the Purkinje cell layer of cerebellum beginning shortly after birth. By P5 the entire layer was positive and signal in this region could be localized to Bergmann glia by P15. A transient elevation in GFAP mRNA was apparent during the second postnatal week in cerebellum and cerebrum. Using in situ hybridization, a peak in message levels was observed at P15 and could be localized primarily to the deep white matter of cerebellum, to the corpus callosum, and to certain hippocampal fiber tracts. The pattern of GFAP expression in these regions is consistent with the differentiation of interfascicular glia and the appearance of type-2 astrocytes during the initial events of myelination. GFAP mRNA levels in white matter were greatly reduced in the adult. The pronounced regional differences in GFAP mRNA expression during development may reflect the differentiation of subpopulations of astrocytes.
Aging↗
Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective.
Is the heat shock response physiologically relevant? For example, following hyperthermia or ischemia, what neural cell types show induction of heat shock genes and what is the time course of the effect? Initial experiments in this area demonstrated the prominent induction of a 70 kDa heat shock protein (hsp70) when labeled brain proteins isolated from hyperthermic animals were analyzed. Recently, in situ hybridization and immunocytochemistry have been utilized to map out the pattern of expression of both constitutively expressed and stress-inducible members of the hsp70 multigene family. Different types of neural trauma have been found to induce characteristic cellular responses in the mammalian brain with regard to the type of brain cell that responds by inducing hsp70 and the timing of the induction response. Fever-like temperature causes a dramatic induction of hsp70 mRNA within 1 hr in fiber tracts of the forebrain and cerebellum, a pattern consistent with a strong glial response to heat shock. Tissue injury, namely, a small surgical cut in the cerebral cortex, induces a rapid and highly localized induction of hsp70 mRNA in cells proximal to the injury site. Using an immunocytochemical approach, a neuronal pattern of induction of hsp70 has been demonstrated following ischemia or kainic acid-induced seizures. It is apparent that the pattern of induction of hsp70 may be a useful early marker of cellular injury and may identify previously unrecognized areas of vulnerability in the nervous system.
Animals↗
Time course of induction of a heat shock gene (hsp70) in the rabbit cerebellum after LSD in vivo: involvement of drug-induced hyperthermia.
In situ hybridization studies were carried out to determine whether induction of hsp70 mRNA in various cellular layers of the rabbit cerebellum was due to hyperthermic effects of the psychotropic drug LSD. Results indicated that induction was not present when LSD-induced hyperthermia was blocked. The pattern of induction of hsp70 mRNA in various cell types of the cerebellum was similar when hyperthermia was induced by either drug (LSD) or nondrug means (placement of animals in a warm incubator). A time course analysis of the induction of hsp70 mRNA following LSD-induced hyperthermia revealed maximal levels of mRNA at 1 hr in all cerebellar cell layers except the Purkinje layer where highest levels were attained at 5 hr. By 10 hr hsp70 mRNA had returned to constitutive levels in all cellular layers of the cerebellum.
Animals↗
Induction of a heat shock gene (hsp70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue.
Elevation of body temperature by 2-3 degrees C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.
Animals↗
Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC.
We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.
Activated-Leukocyte Cell Adhesion Molecule↗
Expression of the beta-S100 gene in brain and craniofacial cartilage of the embryonic rat.
The distribution of beta-S100 mRNA within the brain and skull of the 14-day rat embryo was determined using in situ hybridization. In nonnervous tissue, signal corresponding to beta-S100 mRNA was prominent within cartilaginous regions. Signal appeared to be confined to chondrocytes and was not observed within undifferentiated cells of the perichondrium. Within the skull, expression of beta-S100 mRNA was prominent in the nasal region and maxillary process as well as within Meckel's cartilage. Signal was also observed within the cranial base of the embryonic skull. In the embryonic brain, beta-S100 mRNA was prominent within the ventricular region of the myelencephalon and could be localized to a layer of cells lining the ventricle. Although beta-S100 mRNA has been shown to accumulate postnatally in the central nervous system, the present results clearly identify high levels of beta-S100 mRNA within specific regions of the embryonic brain and skull.
Animals↗
Induction of stress (heat shock) protein 70 and its mRNA in rat corneal epithelium by hyperthermia.
Because stress proteins are believed to play an important role in cellular repair and survival mechanisms, we investigated accumulation of the 70-kilodalton stress protein (SP70) and its mRNA in the rat corneal epithelium after hyperthermia. In the corneal epithelium of control rats, in situ hydridization with a radioactive probe for SP70 mRNA followed by autoradiography revealed very few silver grains. Eighteen hours after the rats were subjected to hyperthermia, the density of silver grains was greatly increased and this elevated level of corneal epithelium SP70 expression continued through 50 hours after heat treatment. Immunostaining for SP70 in the corneal epithelium was consistent with the in situ hybridization, being weak and mainly confined to the basal cells in control rats and increasing by 18 hours in the heat-treated rats. At 50 hours post-heat treatment, the immunostaining was denser than control in all corneal epithelial cells, especially in the apical portions of the basal and wing cells. These results suggest that SP70 may be an important factor in the response of the corneal epithelium to adverse environmental changes.
Animals↗
Cellular localization of heat shock gene expression in rabbit cerebellum by in situ hybridization with plastic-embedded tissue.
The major cell types in rabbit cerebellum which engage in the expression of a heat shock gene (hsp70) after hyperthermia were identified. This required in situ hybridization on thin sections derived from plastic-embedded tissue. All classes of cerebellar neurons which were examined (Purkinje, granule, and stellate cells) responded by induction of hsp70 mRNA within 1 hr after hyperthermia. Prominent induction of hsp70 mRNA was also observed in oligodendroglia in the deep white matter.
Animals↗
Genes expressed in cortical neurons--chromatin conformation and DNase I hypersensitive sites.
DNase I sensitivity experiments were performed utilizing DNA probes to genes which are either transcribed in rat cortical neurons (the 68 kDa neurofilament gene and the neuron-specific enolase gene) or are transcriptionally silent (albumin). Results suggest that unlike liver, in which a hierarchy in chromatin conformation exists between transcribed and nontranscribed genes, the majority of protein coding sequences in cortical neurons may be relatively sensitive to nuclease digestion. This supports our previous observation of an increased DNase I sensitivity of total chromatin in cortical neurons. Nuclease sensitivity experiments also revealed the presence of brain-specific DNase I hypersensitive sites associated with the two neuron-specific genes.
Aging↗
Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischemia.
Rats aged 7 days were exposed to 3.5 h of cerebral hypoxia-ischemia produced by unilateral common carotid artery ligation combined with hypoxia (8% oxygen). The major inducible heat-shock protein, HSP-68, was synthesized in ipsilateral but not contralateral (control) hippocampus during early recovery (1 and 3 h). HSP-68 synthesis was not detected during longer recovery periods. The presence of HSP-68 was confirmed by Western blotting and immunostaining with a polyclonal antibody to HSP-68.
Animals↗