Female genital mutilation in Britain.
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Biomedical subjects
J A Black
Publications and source records attributed to J A Black.
Isoform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations.
The expression of sodium channel alpha-subunit isoforms in astrocytes cultured from P-0 rat spinal cord and P-7 rat optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and isoform-specific amino acid sequences of the rat brain sodium channel. In spinal cord cultures at 5 days in vitro (DIV), both stellate and flat astrocytes were immunostained with antibody SP20, which recognizes a conserved sequence common to sodium channel types I, II/IIA, and III. Antibody SP11-I, which is directed against a subtype-specific sequence in sodium channel I, did not yield detectable staining in spinal cord astrocytes. Antibody SP11-II, which is directed against a subtype-specific sequence in sodium channel II, immunostained both stellate and flat spinal cord astrocytes, although with less intensity than SP20. Antibody SP32-III, which is directed against a subtype-sequence in sodium channel III, immunostained stellate but not flat spinal cord astrocytes. SP20, SP11-II, and SP32-III staining persisted in stellate spinal cord astrocytes through 14-21 DIV, while SP20 and SP11-II immunostaining in flat spinal cord astrocytes was attenuated with time in culture. In optic nerve cultures at 5 DIV, SP20 staining was present in both stellate and flat astrocytes, but at reduced levels compared to spinal cord astrocytes. With increased time in culture SP20 staining was maintained in stellate optic nerve astrocytes but was gradually lost in flat optic nerve astrocytes. Stellate optic nerve astrocytes exhibited low levels of staining with SP11-I, SP11-II, and SP32-III. Flat optic nerve astrocytes lacked or displayed very low SP11-II staining, and SP11-I and SP32-III staining was not detectable. These observations demonstrate that cultures astrocytes are immunoreactive to antibodies generated against conserved and isotype-specific peptide sequences of rat brain sodium channels, and further suggest that there are different patterns of sodium channel expression between flat vs. stellate astrocytes and in astrocytes derived from different regions of the CNS.
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Expression of sodium channel alpha- and beta-subunits in the nervous system of the myelin-deficient rat.
Using subtype-specific riboprobes and a non-isotope in situ hybridization technique, the pattern of expression of the mRNAs for voltage dependent sodium channel alpha-subunits I, II, III and NaG, and the beta 1-subunit were compared in myelin-deficient rats and unaffected male littermates. Tissues examined included the hippocampus, cerebellum, spinal cord and dorsal root ganglia. Previous studies have demonstrated that the expression of sodium channel alpha- and beta 1-subunits follows a distinct temporal and spatial pattern during development, characterized in part by greater expression of alpha-subunit III and its mRNA during development than in the adult. We examined animals of 20-22 days of age, a time when, according to earlier reports, the unaffected animals should nearly have reached an adult expression pattern. Normal male littermates were indeed found to express a sodium channel subunit mRNA pattern generally consistent with previous reports on adult rats. Myelin-deficient animals exhibited an expression pattern identical to the unaffected littermates, indicating that myelination is not required for the progression from the embryonic to the adult expression pattern of sodium channel subunits.
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Differential up-regulation of sodium channel alpha- and beta 1-subunit mRNAs in cultured embryonic DRG neurons following exposure to NGF.
Although the pattern of expression of various sodium channel alpha- and beta-subunits changes as development proceeds, the mechanisms that control the expression of these subunits are not yet understood. To study the role of nerve growth factor (NGF) in modulating the expression of sodium channel subunits, we used in situ hybridization cytochemistry to examine the distribution of sodium channel alpha- and beta 1-subunit mRNAs in embryonic day 16 (E16) dorsal root ganglia (DRG) neurons cultured in the absence or presence of NGE. At 4 days in vitro in the absence of NGF, sodium channel alpha-subunit II mRNA was expressed at low-to-moderate levels in DRG neurons, but the transcripts for sodium channel alpha-subunits I, III and NaG and beta 1-subunit were not detectable. In the presence of NGF, DRG neurons expressed low-to-moderate levels of sodium channel alpha-I, high levels of alpha-II and low levels of alpha-III; NaG mRNA was not detectable. Sodium channel beta 1 mRNA was up-regulated and was expressed at high levels in DRG neurons in NGF-containing media. These observations demonstrate that the NGF exerts a differential up-regulation of sodium channel alpha- and beta-subunit mRNAs in DRG neurons derived from E16 embryos.
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Na+ channel beta 1 subunit mRNA: differential expression in rat spinal sensory neurons.
The brain Na+ channel beta 1 subunit (Na beta 1) mRNA has recently been localized within rat central nervous system where it is expressed at differing levels in different types of neurons. In the present study, we have studied the expression pattern of Na beta 1 mRNA in rat dorsal root ganglion (DRG) neurons using non-radioactive in situ hybridization histochemistry. Na beta 1 mRNA is differentially expressed in adult DRG, with higher levels in intermediate-to-large (> approximately 25 microns in diameter) DRG neurons than in small (< 25 microns) DRG neurons. This cell body size-related Na beta 1 mRNA expression is consistently observed beginning at postnatal day 4 and continues throughout development to adulthood. The present results indicate that (i) Na beta 1 mRNA is expressed in neurons in the peripheral nervous system and (ii) Na beta 1 gene expression is differentially regulated in DRG neurons in relation to their cell body sizes.
Animals↗
Diarrhoea, vitamin K, and warfarin.
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Diarrhea↗
Rat brain Na+ channel mRNAs in non-excitable Schwann cells.
The expression of rat brain voltage-sensitive Na+ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na+ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na+ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.
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Structure and mechanism of interleukin-1 beta converting enzyme.
Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.
Amino Acid Sequence↗
Anoxic injury of rat optic nerve: ultrastructural evidence for coupling between Na+ influx and Ca(2+)-mediated injury in myelinated CNS axons.
Physiological studies in the anoxic rat optic nerve indicate that irreversible loss of function, measured by the compound action potential, is due to depolarization and run-down of the transmembrane Na+ gradient which triggers Ca2+ entry through reverse Na(+)-Ca2+ exchange. EM studies in the anoxic optic nerve have demonstrated characteristic changes, including mitochondrial swelling and dissolution of cristae, submyelinic vacuoles, detachment of perinodal oligodendrocyte-axon loops, and severe cytoskeletal damage with loss of microtubules and neurofilaments within the axoplasm. To further examine the coupling between Na+ influx and Ca(2+)-mediated injury in myelinated axons within anoxic white matter, we have examined the ultrastructural effects of tetrodotoxin (TTX), in the anoxic optic nerve. Optic nerves, maintained in an interface brain slice chamber, were exposed to a 60-min period of anoxia. TTX (1 microM) was introduced 10 min before the onset of anoxia. Nerves were examined at the end of the anoxic period, or after 80 min in 1 microM TTX for normoxic controls. Under normoxic conditions, optic nerve axons exposed to TTX exhibited a normal ultrastructure. In optic nerves exposed to TTX studied at the end of a 60-min period of anoxia, mitochondria showed swelling and loss of cristae, and terminal oligodendroglial loops were detached from the nodal axon membrane. Cytoskeletal architecture was preserved in anoxic optic nerve axons treated with TTX, and axonal microtubules and neurofilaments maintained their continuity. Submyelinic empty spaces were not present. Perinodal astrocyte processes often appeared to be replaced by cellular remnants containing multiple membranous profiles; clusters of shrunken astrocytic processes were present between myelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)
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B2 bradykinin receptors in NG108-15 cells: cDNA cloning and functional expression.
Two distinct cDNAs encoding bradykinin receptors (BKRs) were cloned from NG108-15 neuroblastoma-glioma hybrid cells. One was identical with rat uterus B2 BKR, whereas the other one (mBKR) had 91% amino acid homology to the rat B2 BKR and 82% homology to human B2 BKR. Southern blot analysis and genomic DNA cloning revealed that mBKR is derived from the mouse genome. The mBKR, expressed in Xenopus oocytes and COS-7 cells, produced functional BKRs that exhibited the properties of smooth muscle type B2 BKR. These results suggest that both the rat and mouse B2 BKRs of the smooth muscle type are expressed in NG108-15 cells.
Amino Acid Sequence↗
Type II sodium channels in spinal cord astrocytes in situ: immunocytochemical observations.
The expression of sodium channel alpha-subunit isoforms in astrocytes in adult rat spinal cord and optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and subtype-specific sequences of the sodium channel. In adult rat spinal cord, astrocytes within the dorsal and ventral funiculi were immunolabelled with antibody SP20, which recognizes a conserved sequence within sodium channel types I, II, and III. In addition, astrocytes within these spinal cord white matter tracts were immunostained with antibody SP11-II, which recognizes sodium channel type II. Antibodies SP11-I and SP32-III, which are directed against subtype-specific sequences in sodium channel types I and III, respectively, did not label astrocytes in the dorsal and ventral funiculi of the spinal cord. In optic nerves, astrocytes were immunostained with antibody SP20. However, no detectable labelling of cells within the optic nerve was observed with antibodies SP11-I, SP11-II, and SP32-III. These observations demonstrate that sodium channel II is expressed by astrocytes in spinal cord white matter. Moreover, these data suggest that regional factors regulate the level of sodium channel isoform expression in astrocytes.
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Sodium channel mRNAs I, II and III in the CNS: cell-specific expression.
The cellular localization of rat brain sodium channel alpha-subunit mRNAs I, II and III in the central nervous system (CNS) was examined by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes, one set recognizing sodium channel isoforms in the coding region and the other in the non-coding region of the sodium channel messages. The independent sets of probes demonstrated qualitatively similar patterns of sodium channel mRNA expression. In the hippocampus, sodium channel mRNA I was very weakly expressed in the pyramidal layer and in the granular layer of the dentate gyrus; in contrast, sodium channel mRNA II was strongly expressed by neurons in these regions. Sodium channel mRNA III exhibited low-to-moderate expression in some neurons of the pyramidal layer of the hippocampus and granular layer of the dentate gyrus, and was not detectable in others. In the cerebellum, sodium channel mRNA I was moderately expressed in some Purkinje cells, weakly expressed in scattered cells in the molecular layer and negligibly expressed in the granular layer. Sodium channel mRNA II was strongly expressed in Purkinje and granule cells, and was moderately expressed in some cells in the molecular layer. Sodium channel mRNA III was generally not detectable in the cerebellum. In the spinal cord, motor neurons and scattered neurons throughout the gray matter exhibited moderate-to-strong expression of both sodium channel mRNA I and II. A population of cells in the spinal zone of Lissauer showed heavy expression of mRNA II, but not mRNA I. Sodium channel mRNA III was not detectable in spinal cord neurons. These observations are consistent with a general regional distribution of sodium channel message isoforms, with mRNA II being preferentially expressed in rostral regions of the CNS and mRNA I in caudal regions. However, the results also indicate that different cell types, within a given region, display different patterns of sodium channel mRNA expression. Moreover, these data suggest that individual neurons may express multiple forms of sodium channel mRNA.
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The expression of rat brain voltage-sensitive Na+ channel mRNAs in astrocytes.
Astrocytes from various regions of CNS have been shown to express voltage-activated Na+ currents. To date, three distinct subtypes (I, II and III) of Na+ channels have been cloned from rat brain. We have applied a combined technique of reverse transcription and polymerase chain reaction (RT-PCR) to examine the expression of rat brain Na+ channels in rat astrocytes in vivo and in vitro. Five PCR primer sets were used to amplify coding or 3' non-coding regions of subtype I, II, and III Na+ channels. We were able to amplify all three of these rat brain Na+ channel subtypes from rat optic nerve, which does not have neuronal cell bodies but does contain astrocytes known to express voltage-sensitive Na+ channels. In studies on cultured spinal cord astrocytes, we were also able to amplify all three subtypes of rat brain Na+ channel mRNAs. In control experiments, RT-PCR was performed on RNAs prepared from several rat tissues, including brain, skeletal muscle, and liver. Rat brain was shown to express the three Na+ channel subtypes as expected. In rat skeletal muscle, subtype I and III Na+ channel mRNAs, but not subtype II, were amplified. In rat liver, Na+ channel messages were not detectable. The present study provides the first direct evidence that astrocytes in vivo and in vitro express rat brain voltage-sensitive Na+ channel mRNAs, which have been considered as mainly neuronal-type Na+ channel messages.
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Sodium channel mRNAs in cultured spinal cord astrocytes: in situ hybridization in identified cell types.
The expression of rat brain sodium channel alpha-subunit mRNAs I, II and III and a putative glial cell-specific sodium channel (NaG) mRNA was examined in cultured astrocytes from P-0 rat spinal cord by RNA blot hybridization and by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes. Sodium channel mRNA I was not detectable in the cultured astrocytes by RNA blot or in situ hybridization. Sodium channel mRNA II showed negligible-to-low levels of expression in flat, fibroblast-like and 'pancake' astrocytes at 4 days in vitro (div), while stellate, process-bearing astrocytes exhibited low-to-moderate levels of mRNA II expression. At 7 div, mRNA II expression ranged from low-to-moderate in flat astrocytes and was moderately high in most process-bearing astrocytes. In RNA blots, a weak band was observed at 9.5 kb. Sodium channel mRNA III expression was negligible in flat astrocytes and was detectable in low-to moderate levels in stellate astrocytes beginning at 4 div; by 7 div, mRNA III was detectable in low levels in flat astrocytes and low-to-moderate levels in stellate astrocytes. RNA blots showed two bands of nearly equal intensity, one at 9.0 kb and one at 7.2 kb. NaG mRNA showed increased expression with time in culture, being detectable in flat and stellate astrocytes at 4 div and becoming very prominent in flat astrocytes at extended times in culture. In RNA blots of cultured astrocytes at 7 div, a strong hybridizing signal with the NaG probe was observed. These observations demonstrate that flat and stellate astrocytes cultured from rat spinal cord express rat brain sodium channel mRNA II and III, and NaG, and suggest that astrocytes in vitro may co-express multiple forms of sodium channel mRNA.
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Type III sodium channel mRNA is expressed in embryonic but not adult spinal sensory neurons, and is reexpressed following axotomy.
1. In situ hybridization with subtype-specific probes was used to ask whether there is a change in the types of sodium channels that are expressed in dorsal root ganglion (DRG) neurons after axotomy. 2. Types I and II sodium channel mRNA are expressed at moderate-to-high levels in control DRG neurons of adult rat, but type III sodium channel mRNA is not detectable. 3. When adult rat DRG neurons are examined by in situ hybridization 7-9 days following axotomy, type III sodium channel mRNA is expressed at moderate-to-high levels, in addition to types I and II mRNA that are present at relatively high levels. 4. To determine whether the expression of type III sodium channel mRNA following axotomy represents up-regulation of a gene that had been expressed at earlier developmental stages, we also studied DRG neurons from embryonic (E17) rats. In these embryonic DRG neurons, type I sodium channel mRNA is expressed at low levels, type II mRNA at high levels, and type III at high levels. 5. These results demonstrate altered expression of sodium channel mRNA in DRG neurons following axotomy, and suggest that in at least some DRG neurons, there is a de-differentiation after axotomy that includes a reversion to an embryonic mode of sodium channel expression. Different channel characteristics, as well as an altered spatial distribution of sodium channels, may contribute to the electrophysiological changes that are observed in axotomized neurons.
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Glial cells and axo-glial interactions: implications for demyelinating disorders.
Two major glial cell types, the oligodendrocyte (which produces myelin) and the perinodal astrocyte (which contacts the sodium channel-rich axon membrane at the node of Ranvier), collaborate with the axon in forming the central myelinated fiber. Astrocytes have been demonstrated to synthesize voltage-sensitive sodium channels, and in some astrocytes these channels have neuronal properties. A variety of lines of evidence suggest that astrocytes play an important role in the reorganization of the axon membrane following demyelination. Possible functions of astrocytes include the targeting, or anchoring, of ion channels at specific sites within the axon membrane, or the specification of channel characteristics as a result of production of extracellular matrix molecules that interact with the glycosylated dome of sodium channels, or the synthesis of channels that are subsequently transferred to axons. It is also possible that astrocytes participate in ionic homeostasis at the node of Ranvier. A second major glial cell type, the oligodendrocyte, produces myelin during normal development. Morphologic studies have demonstrated that transplantation of myelin-forming cells, or their precursors, can result in the production of myelin with normal structure in the myelin-deprived CNS. In recent physiological studies, the function of axons following myelination by exogenous, transplanted glial cells has been examined. In regions of glial cell transplantation, there are significant increases in conduction velocities that approach normal values. Both of these lines of investigation, on astrocytes and myelin-forming oligodendrocytes/oligodendrocyte precursors, suggest that manipulation of glial cell properties may emerge as a viable strategy for promoting the recovery of conduction in CNS white matter axons following demyelination.
Action Potentials↗
Protection of the axonal cytoskeleton in anoxic optic nerve by decreased extracellular calcium.
Since CNS white matter tracts contain axons, oligodendrocytes and astrocytes but not synapses, it is likely that anoxic injury of white matter is mediated by cellular mechanisms that do not involve synapses. In order to test the hypothesis, that anoxic injury of white matter is mediated by an influx of Ca2+ into the intracellular compartment of axons, we compared the ultrastructure of axons in rat optic nerve exposed to 60 min of anoxia in artificial cerebrospinal fluid (aCSF) containing normal (2 mM) Ca2+, and in aCSF containing zero-Ca2+ together with 5 mM EGTA. Optic nerves fixed at the end of 60 min of anoxia in 2 mM Ca2+ exhibit extensive ultrastructural alterations including disruption of microtubules and neurofilaments within the axonal cytoskeleton, development of membranous profiles and empty spaces between the axon and the ensheathing myelin, and swelling of mitochondria with loss of cristae. Bathing the nerves in zero-Ca2+ aCSF during anoxia protected the axons from cytoskeletal changes; after 60 min of anoxia, optic nerve axons retained normal-appearing microtubules and neurofilaments. Membranous profiles were rare, and empty spaces between axons and myelin did not develop in anoxic optic nerves bathed in zero-Ca2+ aCSF. Disorganization of cristae in axonal mitochondria was observed in anoxic optic nerves even when Ca2+ was omitted from the medium. Because Ca(2+)-mediated injury is known to disrupt the axonal cytoskeleton, these results support the hypothesis that anoxia triggers an abnormal influx of Ca2+ into myelinated axons in CNS white matter.
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Spinal cord astrocytes in vitro: phenotypic diversity and sodium channel immunoreactivity.
The expression of sodium channels in morphologically and antigenically distinct astrocytes derived from neonatal rat spinal cords was examined at various times in culture. During the course of this study [2-40 days in vitro (DIV)], nine morphologies of glial fibrillary acidic protein (GFAP)+ cells were distinguished: 1a) flat, fibroblast-like; 1b) elongated, with generally few, short processes; 1c) triangular soma with three short, stubby processes; 1d) bipolar with long, slender processes; 1e) bipolar with broad, flared processes; 1f) stellate with radially oriented slender processes extending from a small to moderate-sized soma; 1g) multiple short, stubby processes extending from a moderate-sized soma; 1h) flat, roundish shape with either a smooth edge ("pancake"-like) or numerous very short processes; and 1i) broad, elongated cell body with orthogonally oriented short, spike-like processes. Not all cell types were present at all times in culture. Each type of astrocyte displayed sodium channel immunoreactivity at some time in culture; however, different types of astrocytes exhibited different patterns, over time, of sodium channel staining. Sodium channel immunoreactivity in all astrocyte types was reduced to low levels by 14 DIV, and was not detectable at 40 DIV. Except for types 1b and 1e, A2B5 staining was present on all astrocyte morphologies at some time in culture, and was generally attenuated with longer times in vitro; in contrast to cultures derived from neonatal rat optic nerve, A2B5 staining does not distinguish unequivocally between the various classes of morphologically different astrocytes derived from spinal cord. O4 immunoreactivity was consistently observed only on bipolar, elongated, and process-bearing astrocytes, though not all process-bearing astrocytes were O4+. These results demonstrate that astrocytes derived from neonatal spinal cord are morphologically and antigenically heterogeneous. Moreover, while spinal cord astrocytes express sodium channels, these astrocytes exhibit a time-course of channel expression that is different from astrocytes derived from several other CNS regions where sodium channel staining is maintained even for extended times in culture, suggesting a regional modulation of astrocyte function.
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