Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development

Adult adrenal chromaffin cells are being utilized for therapeutic transplantation. With the prospect of using fetal chromaffin cells in pain therapy, we studied their phenotype, proliferative power, function, and growth in vitro and in situ in order to determine the optimal time for implantation. Between 7 and 10 gestational weeks (GW), we isolated, in vitro, two types of chromaffin cells with a noradrenergic phenotype akin to that observed, in situ. Among the adherent chromaffin cells first observed in vitro, only a few samples expressed met-enkephalin, whereas almost all the neurosphere-like colonies, which appeared later, expressed it. However, neither of the two types of populations expressed an adrenergic phenotype in line with that observed in situ. At the upper limits of the voluntary abortion period authorized in France, this phenotype (12 GW) and met-enkephalin expression (13 GW) were evidenced in situ. For the first time in man, we demonstrate the secretion of noradrenaline in vitro by the two populations of cells. Consistent with this result, we also noted dopamine beta hydroxylase (DbetaH) mRNA expression in vitro and in situ within this period. These observations on the expression of these biological factors indicate that 9-10 GW would be the best stage for sampling these cells for preclinical transplantation experiments.     

Human fetal chromaffin cells: a potential tool for cell pain therapy

Transplantation of adrenal medulla cells has been proposed in the treatment of various conditions. Indeed, these cells possess a bipotentiality: neural and neuroendocrine, which could be exploited for brain repair or pain therapy. In a previous study, we characterized these human cells in vitro over 7-10 gestational weeks (GW) [Zhou, H., Aziza, J., Sol, J.C., Courtade-Saidi, M., Chatelin, S., Evra, C., Parant, O., Lazorthes, Y., and Jozan, S., 2006. Cell therapy of pain: Characterization of human fetal chromaffin cells at early adrenal medulla development. Exp. Neurol. 198, 370-381]. We report here our results on the extension to 23 GW. This developmental period can be split into three stages. During the first stage (7-10 GW), we observed in situ that extra-adrenal surrounding cells display the same morphology and phenotype as the intra-adrenal chromaffin cells. We also found that the intra-adrenal chromaffin cells could be committed in vitro towards an adrenergic phenotype using differentiating agents. During the second stage (11 to 15-16 GW), two types of cells (Type 1 and Type 2 cells) were identified morphologically both inside and outside the gland. Interestingly, we noted that the Type 2 cells stem from the Type 1 cells. However, during this developmental period only the intra-adrenal Type 2 cells will evolve towards an adrenergic phenotype. In the third stage (17-23 GW), we observed the ultimate location of the medulla gland. Both the in situ results and the in vitro experiments indicate that particular procedures need to be implemented prior transplantation of chromaffin cells. First, in order to obtain a large number of immature chromaffin cells, they must be isolated from the intra and extra-adrenal gland and should then be committed towards an adrenergic phenotype in vitro for subsequent use in pain therapy. This strategy is under investigation in our laboratory.     

Unfavorable outcome of adrenal medullary transplant for Parkinson's disease

Summary A 45-year-old woman with moderately severe parkinsonism underwent adrenal medullary autograft to the brain. The operation was performed in Mexico City. There were multiple post-Operative complications, including worsening of the parkinsonism with almost complete loss of ability to communicate. Death from bronchopneumonia occurred 13 months after surgery. At autopsythe surgical tract was found to extend through the corpus callosum and into the thalamus on the oppsiite side. Residual necrotic adrenal tissue was found as a small round well-defined mass in the ventricle loosely attached to the left fornix. This case emphasizes the obvious risks of this still experimental procedure and the difficulties in obtaining a surviving and functional adrenal medullary graft.Supported by the Veterans Affairs medical Research Program and the California Parkinson's Foundation     

Implantation of cultured human leptomeningeal cells into rat brain

Since previous studies have shown that cells cultured from human leptomeninges can express neuronal and glial antigens under appropriate culture conditions [DeGiorgio L. A. et al. (1994) J. Neurol. Sci.124, 141–148; Bernstein J. J. et al. (1996) Int. J. Devl Neurosci.14(5), 681–687], we have studied the developmental characteristics of these cells further by grafting them into young adult rat brains. Cells were labeled in culture with Fast Blue and were identified unequivocally by hybridization with nick-translated human DNA. Intensely Fast Blue positive human leptomeningeal cells were concentrated in the implant pocket and adjacent rat leptomeninges at one and two weeks postimplant. Human and rat leptomeningeal cells were similar morphologically and were equally immunopositive for vimentin and fibronectin. Implanted human cells did not express the neuronal and glial proteins they had in vitro. Cells which hybridized with human DNA corresponded to the intensely Fast Blue positive cells. Small groups of human DNA hybridizing cells were also observed in the choroid plexus. Less intensely Fast Blue positive neurons and glia were found in the brain, but these hybridized with rat DNA. A minority of human leptomeningeal cells implanted into rat brain are subsequently found in host leptomeninges where they demonstrate properties characteristic of leptomeningeal fibroblasts. Small numbers of implanted cells can survive for two weeks.     

Transplantation of adrenal tissue into the central nervous system

Adrenal medullary tissue can survive transplantation to the central nervous system. Such survival has been obtained experimentally with grafts to the anterior eye chamber, to the brain and to the spinal cord, using medullary tissue from the recipient animal or unrelated animals of the same or, in some cases, different species. Appropriately placed grafts have been shown, under certain conditions, to interact with the host nervous system, exerting behavioral effects including amelioration of experimentally-induced parkinsonian symptoms. Such effects may be enhanced by administration of nerve growth factor to the grafts. On the basis of such findings, adrenal medullary tissue has been grafted to the brain of Parkinson's disease patients. Both animal and human experiments raise important questions about mechanisms of graft action and about factors that influence the outcome of these procedures.     

Actions of somatostatin on perfused bovine adrenal glands and cultured bovine adrenal medullary cells

The effects of somatostatin on catecholamine secretion and inositol phosphate accumulation have been studied using isolated perfused bovine adrenal glands and cultured bovine adrenal medullary cells. Somatostatin had no effect on basal adrenaline or noradrenaline secretion from either preparation. At concentrations above 1 microM, somatostatin inhibited the secretion of both catecholamines induced by 5 microM nicotine from cultured chromaffin cells. In contrast, over the concentration range 0.1 nM-10 microM, somatostatin had no effect on the secretory responses produced by 10 nM angiotensin II or 1 microM histamine. Inositol phosphate accumulation in cultured bovine adrenal medullary cells was unaffected by 0.1 nM-0.1 microM somatostatin, however at 1 and 10 microM somatostatin it was significantly increased, by 23% and 103% respectively. The effects of somatostatin (0.1 nM-10 microM) and of 50 microM muscarine on inositol phosphate accumulation were simply additive. Similarly, somatostatin at 0.1 nM and 10 nM together with 10 nM angiotensin II or 1 microM histamine produced additive inositol phosphate responses. In contrast, 1 microM somatostatin gave significantly more-than-additive (synergistic) inositol phosphate responses with angiotensin II and histamine. The results suggest that some adrenal medullary cells possess several types of receptors, and that these receptors may interact to produce non-additive responses.     

Functional recovery in a rat model of Parkinson's disease following transplantation of cultured human sympathetic neurons

Rats subjected to a unilateral 6-hydroxydopamine (6-OHDA)-induced lesion of the nigostriatal dopamine pathway were given transplants of cultured fetal human sympathetic neurons. Amphetamine-induced turning behavior in these rats was reversed by the transplants after 1.5–4.5 months. The presence of transplanted neurons and their processes was demonstrated by immunohistochemistry for tyrosine hydroxylase.     

Low power laser irradiation enhances migration and neurite sprouting of cultured rat embryonal brain cells

Abstract

We used fetal brain cells grown in tissue culture to study some basic features of the interaction between low-power laser irradiation and biological systems. Seven- to nine-day-old rat fetal brain cell aggregates in culture were subjected to direct focused irradiation of low-power helium-neon laser (0.3 mV\f, 632.8 nm). An 8-minute dose of laser irradiation enhanced the appearance of brain cells around the treated aggregates, as monitored under the microscope of the stained cultures. Two and three doses of laser irradiations were correlated with 97% and 142% respective increases of the numbers of cells surrounding the aggregates. To identify the type of cells grown in the outgrowth of the treated aggregate> specific tetanus-anti-tetanus antibodies were used. Rhodamine-labeled antibodies bound to receptors on cells indicated massive neurite sprouting and outgrowth of migrating brain cells in culture. [Neurol Res 1996; 18: 467-470]     

Ultrastructural and cytochemical characteristics of cultured rat Schwann cells

Summary Schwann cell cultures were established from sciatic nerve of 3 day-old rats. Described are the ultrastructural, histochemical and ultracytochemical properties of amyelic cultured rat Schwann cells. Ultrastructural characteristics of the cultured Schwann cells are compared to the Schwann cells of 3 day-old and adult rat sciatic nerve. These findings serve as a basis for comparison when studying experimentally induced alterations in the cultured Schwann cells as well as changes due to myelination in vitro.     

Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.     

Vasoactive intestinal peptide potentiates and directly stimulates catecholamine secretion from rat adrenal chromaffin cells

The actions of vasoactive intestinal polypeptide (VIP) on catecholamine secretion and changes in [Ca²⁺]_i in single rat chromaffin cells were studied using amperometry and Indo-1. Application of VIP prior to acetylcholine (ACh) or co-application of VIP and ACh enhanced secretion by 94% and 153% respectively, compared to ACh alone. [Ca²⁺]_i was increased by 17% when VIP was preapplied and by 73% upon co-application. Exposure to VIP before stimulation with 60 mM K⁺ enhanced secretion by 68%, but not [Ca²⁺]_i. VIP application prior to DMPP and nicotine had no effect on [Ca²⁺]_i, but increased [Ca²⁺]_i signals to muscarine by 18%. VIP co-application potentiated only [Ca²⁺]_i responses to muscarine, by 28%. The effect of VIP on muscarine-induced [Ca²⁺]_i signals was mimicked by 8-Br-cAMP, and both were blocked by H-89, a protein kinase A inhibitor. Long-lasting increases in secretion accompanied by a sustained rise in [Ca²⁺]_i to VIP alone were seen in 55% of cells. Removal of Ca²⁺ or addition of La³⁺ inhibited both responses, while L-, N- and P-type Ca²⁺ channel blockers were ineffective. SK&F 96365 inhibited VIP-induced secretion completely and rises in [Ca²⁺]_i by 75%. Neither 8-Br-cAMP nor 8-Br-cGMP evoked responses similar to VIP alone. Thus in rat chromaffin cells, VIP acts both directly as a neurotransmitter in provoking sustained catecholamine secretion in a cAMP-independent manner, and also by enhancing ACh-induced secretion, via a cAMP-dependent action involving muscarinic receptors.     

Transplantation of porcine umbilical cord matrix cells into the rat brain

Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were transplanted into the rat brain and recovered in that tissue 2-6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent protein (eGFP) were transplanted. These cells were recovered 2-4 weeks after brain transplantation. Engrafted cells expressing eGFP and positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage.     

Plasma membrane and vesicular glutamate transporter expression in chromaffin cells of bovine adrenal medulla

The study of the functional expression of glutamate signaling molecules in peripheral tissues has received relatively little attention. However, evidence is increasing for a role of glutamate as an extracellular signal mediator in endocrine systems, in addition to having an excitatory amino acid neurotransmitter role in the CNS. Chromaffin cells are good models of catecholaminergic neurons, in which previous work from our group demonstrated the existence of both functional glutamate receptors and specific exocytotic and nonexocytotic glutamate release. In this work, the presence of specific plasma membrane (EAATs) and vesicular glutamate (VGLUTs) transporters has been investigated by using confocal microscopy, flow cytometric analysis, Western blot, and qRT-PCR techniques. We found specific expression of EAAT3, EAAT2, VGLUT1, and VGLUT3 in about 95%, 65%, 55%, and 25%, respectively, of the whole chromaffin cell population. However, chromaffin cells do not express VGLUT2 and have a very low expression of EAAT1. VGLUTs are localized mainly in the membrane fraction, and EAATs share their subcellular location between membrane and cytosolic fractions. Their estimated molecular weights were about 70 kDa for EAAT2, about 65 kDa for EAAT3, about 50 kDa for VGLUT1, and about 60 kDa for VGLUT3. RT-qPCR techniques confirm the expression of these glutamate transporters at the mRNA level and show a different regulation by cytokines and glucocorticoids between VGLUT1 and -3 and EAAT2 and -3 subfamilies. These interesting results support the participation of these glutamate transporters in the process of glutamate release in chromaffin cells and in the regulation of their neurosecretory function in adrenal medulla.     

Observation of cultured peripheral non-neuronal cells implanted into the transected spinal cord

Summary We have previously reported that cultured peripheral non-neuronal cells could be used as an adjunct to spinal cord reconstruction with the delayed nerve graft technique. The cultured cells appeared to enhance axonal regeneration and with their use the time it took for axons from the spinal cord stumps to reach the nerve graft was reduced. To gain insight into the possible mechanisms through which peripheral nonneuronal cells can foster CNS regeneration, we have now investigated the behaviour of the peripheral nonneuronal cells after implantation into the spinal cord. Autologous mixed non-neuronal cell cultures were prepared from cat sciatic nerve biopsies and labeled in culture with tritiated thymidine. The labeled cells were implanted so as to completely fill the gap in the spinal cord produced by a narrow slit transection. Light-and electron-microscopic autoradiography was used to identify the cells 3 and 7 days after implantation and to determine their proximity to, and possible interaction with, axons in the spinal cord stumps. The implanted peripheral cells were frequently found near spinal cord axons and axon terminals. Some of the labeled cells ensheathed axons in which case they displayed morphological characteristics of Schwann cells. Other labeled cells had characteristics of fibroblasts and were surrounded by an extracellular matrix rich in collagen fibrils. Many of the labeled cells contained phagocytosed myelin debris. These observations are consistent with the implanted cells acting to enhance regeneration in the spinal cord either by direct interaction with axons (ensheathment) or indirectly via the production of soluble neuronotrophic factors or a favorable extracellular matrix. The ability of the implanted cells to rapidly move into the spinal cord stumps and attain positions close to spinal cord axons would be an important factor for any of these mechanisms.Supported by grants from the Veterans Administration and the National Institutes of Health (NS-14413)     

Transplantation of human sympathetic neurons and adrenal chromaffin cells into parkinsonian monkeys: no reversal of clinical symptoms

Cultured human fetal sympathetic ganglion explants or adrenal chromaffin cell aggregates were implanted into the left striatum of monkeys whose left nigrostriatal pathway had been lesioned with the neurotoxin MPTP. There was no clinical reversal of parkinsonian symptoms and PET scans did not show increased striatal fluorodopa uptake from pre-implant levels. At sacrifice, left striatal contents of dopamine were not statistically different from MPTP-treated but non-implanted controls. Histological examinations revealed pockets of extrinsic cells which were found at the end of needle tracks. There was no evidence of immune rejection. The extrinsic cells did not stain for tyrosine hydroxylase or neurofilament, suggesting that they were not dopaminergic neurons. The failure to reverse clinical parkinsonian symptoms highlights the stage of infancy of neural implantation in Parkinson's disease.     

Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice

Schwann cells from transected peripheral nerve segments are known to produce nerve growth factor (NGF). We performed adrenal medullary grafts or cografts of adrenal medulla and sciatic nerve into the striatum of MPTP-treated young adult mice, and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and neurochemical analysis with high performance liquid chromatography (HPLC). Adrenal medullary chromaffin cells cografted with sciatic nerve survived better than those in adrenal grafts alone; host DA fiber recovery was more prominent in mice with cografts than in mice with adrenal grafts alone. A large number of TH-IR surviving cells in cografted mice showed long neuronal processes which were rarely seen in the mice receiving adrenal graft alone. We conclude that cograft of adrenal medulla and sciatic nerve promotes intrinsic host DA fiber recovery better than adrenal medulla grafts alone, and that survivability of grafted chromaffin cell may promote host DA fiber recovery. Adrenal medullary autografts have been used in patients with Parkinson's disease; we suggest that if this approach is to be used in the future, methods to increase the survivability of grafted chromaffin cells, such as co-grafting with pieces of peripheral nerve, be considered to enhance the survivability of the chromaffin cells, which might be closely related to the functional recovery of the patients by this grafting procedure. Of course, such strategies as the present cografting approach must be demonstrated to work in older animals using older donor tissue before proceeding to this next step in humans.     

Neural stem cells spontaneously express dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat

The ability to differentiate neural stem cells (NSCs) into dopamine neurons is fundamental to their role in cell replacement therapies for neurodegenerative disorders such as Parkinson's disease. We show here that when a clonal line (C17.2) of undifferentiated NSCs is transplanted into the intact or 6-hydroxydopamine-lesioned striatum, cells withdraw from the cell cycle (BrdU(-)), migrate extensively in the host striatum, and express markers associated with neuronal (beta-tubulin III(+), NSE(+), NeuN(+)) but not glial (GFAP(-), MBP(-), A2B5(-)) differentiation. Importantly, by 2-5 weeks postgrafting, in the majority of these transplants, nearly all engrafted cells express the dopamine-synthesizing enzymes tyrosine hydroxylase and aromatic L-amino decarboxylase, sometimes resulting in changes in motor behavior. In contrast, no NSCs stain for dopamine-beta-hydroxylase, choline acetyltransferase, glutamic acid decarboxylase, or serotonin. We conclude that, following transplantation into the intact or 6-hydroxydopamine-lesioned rat, the adult brain contains intrinsic cues sufficient to direct the specific expression of dopaminergic traits in immature multipotential neural stem cells.     

Effect of substance P on nicotine-induced desensitization of cultured bovine adrenal chromaffin cells: possible receptor subtypes

The neuropeptide substance P (SP) has been reassessed for its ability to modify nicotine-induced catecholamine secretion from cultured bovine, adrenal chromaffin cells. SP exhibited biphasic effects in its actions of inhibiting the nicotinic secretory response and protecting against desensitization. At low concentrations, up to 3 microM, SP partially inhibited or partially protected the nicotine response by 15-20%, and at high concentrations (30 microM), SP markedly inhibited or markedly protected the nicotinic response by 80 or 92%, respectively. The SP antagonist (D-Arg1-D-Pro2-D-Trp7,9-Leu11-SP) completely blocked both effects produced by low concentrations of SP, but not those produced by high concentrations. It is concluded that SP is more potent at protecting against desensitization than at inhibiting the nicotinic response and that SP might modulate CA release through activation of two receptor subtypes.     

Altered reactivity of the rat adrenal medulla

Previous studies have suggested that experimental alterations in adrenomedullary reactivity, i.e., changes in catecholamine release in response to a standard dose of acetylcholine, may be partially accounted for by changes occurring at the level of the adrenal medulla itself, independent of both the central nervous system and the innervation of the adrenal gland. The present study was designed to investigate the morphology of adrenal chromaffin cells in rats subjected to chronic hypoglycemia induced by long acting insulin, and to assess this morphology in terms of associated changes in catecholamine content and release. Surgically isolated, perfused adrenal gland preparations were utilized to characterize the functional release of catecholamines from the adrenal medulla. Pretreatment with long acting insulin resulted in a selective depletion of epinephrine stores and acetylcholine-mediated epinephrine release, but did not appear to significantly affect either the levels or the release of norepinephrine. The biochemical effects of long acting insulin persisted for several days after termination of the treatment, exhibiting a gradual recovery over a period of approximately 5 days. Electron microscopic examination of the adrenal chromaffin cells revealed a progressive degranulation and vacuolization of numerous chromaffin cells followed by a compensatory biosynthetic response and a gradual recovery toward the morphology of control cells. The functional release of catecholamines from adrenal chromaffin cells was further examined in preparations of perfused adrenal slices. Acetylcholine-mediated catecholamine release was significantly decreased in slices of adrenal glands prepared from insulin treated rats when compared with that of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopic localization of S-100 protein in cultured rat glioma cells

Summary Immunoelectron microscopic localization of the nervous system specific protein S-100 in the cultured rat glioma cells was successfully conducted by an unique immunocytochemical technique using peroxidase-labeled antigen binding Fab' fragments.The intensely electron dense reaction product for S-100 protein was localized mainly at ribosome granules associated with endoplasmic reticulum membranes and at free ribosome granules. S-100 protein was also associated to some extent with the cytoplasmic and nuclear membranes. A positive reaction was localized at the nuclear pores as if it were being prevented from entering into the nucleus. No activity was found in the nucleoplasm except for a slightly positive reaction product associated with nucleolus. The possible role for S-100 protein in neural cells was discussed in relation to the nuclear acidic proteins involved in genomic regulation.