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Biomedical subjects

A J Dwork

Publications and source records attributed to A J Dwork.

32 records · Page 2Linked to original sources
PubMed1991

Purification of a nicotinic acetylcholine receptor from rat brain by affinity chromatography directed at the acetylcholine binding site.

We have purified a nicotinic acetylcholine receptor from rat brain by use of an acetylcholine affinity resin commonly employed for the purification of nicotinic acetylcholine receptor from electric tissue. Receptor, specifically eluted with nicotine, bound (-)-[3H]nicotine with a dissociation constant of approximately 21 nM. Binding was inhibited by carbamylcholine but not by alpha-bungarotoxin. Polyacrylamide gel electrophoresis yielded two protein bands, of apparent mol. wts. 80,400 and 52,400. These results provide independent confirmation of the subunit size and composition reported for rat brain nicotinic receptor isolated by immunoaffinity methods and demonstrate a method of purification that can be performed with commercially available reagents.

Acetylcholine↗
PubMed1991

Stereotaxic implantation of autologous adrenal medulla into caudate nucleus in four patients with parkinsonism. One-year follow-up.

Four patients with levodopa-responsive parkinsonism (aged 26, 35, 45, and 49 years) received autologous adrenal medullary implants into or near the left caudate nucleus by stereotaxic implantation after flank adrenalectomy. All patients had an immediate response to implantation lasting several days, during which parkinsonian signs and symptoms decreased. This period was followed by a gradual reappearance of symptoms in all but one patient. This patient had had a dramatic increase in "on" time without dyskinesias and a decrease in the severity and duration of "off" time. He died of multifocal glioblastoma 1 year after transplantation. Autopsy revealed no surviving adrenal cells. In one case, the stereotaxic implantation missed the basal ganglia, resulting in the placement of the adrenal medullary tissue into the medial thalamus and near the third ventricle; the patient did not improve. In the other two cases, a modest but definite increase in "on" time without dyskinesia and a reduction in the severity and duration of "off" time has been observed. The role of autologous adrenal medullary transplantation in patients with parkinsonism remains to be determined. Patients with a family history of cerebral malignancy may be at increased risk for the development of transplant-induced malignancy.

Adrenal Medulla↗
PubMed1991

Autoradiographic study of iron-binding sites in the rat brain: distribution and relationship to aging.

Specific binding of 59Fe to various brain structures was investigated in rats using nanomolar concentrations of 59FeCl3 and quantitative autoradiography. Saturation studies revealed high affinity binding (kd in the nanomolar range) with binding sites density (Bmax) which varied in different brain regions from 462 fmol per mg tissue in the central thalamic nuclei to over 4 pmol per mg tissue in the cerebral peduncle. Binding was seen in both white and gray matter structures. Bmax values for frontal cortex, dentate gyrus, and substantia nigra were significantly lower in older rats. The distribution of 59Fe binding sites was not consistent with the distribution of brain iron as reported by other investigators. 59Fe binding was reduced significantly in the presence of free radicals. These observations suggest that high affinity binding sites for iron are localized differentially in various brain structures and may play an important role in the translocation and storage of potentially harmful ferric cations in brain. The finding that the capacity of the brain tissue to bind iron diminished with age in discrete brain regions suggests that in the aged animal, the removal of "free" iron from the cellular domain may be impaired in such regions, leading to increased susceptibility to iron-enhanced lipid peroxidation and cell death.

Aging↗
PubMed1990

An autoradiographic study of the uptake and distribution of iron by the brain of the young rat.

Rats aged 15, 28, or 42 days were injected intraperitoneally with iron-59 and were sacrificed at varying intervals. Total acquisition of iron-59 by the brain, when compared to levels of iron-59 in blood sampled 48 h after injection, diminished with increasing age at injection. Cerebral levels of iron-59 in animals injected at age 15 days did not change with postinjection interval despite rapidly decreasing serum levels of iron-59. Thus, iron acquired by the brain early in postnatal development becomes sequestered in that organ. However, autoradiography of the brains of animals injected at age 15 days showed definite changes over time in the anatomic distribution of the isotope. This suggests that mechanisms may exist for the translocation of iron from one area of the brain to another.

Aging↗
PubMed1990

The retinal pigment epithelium is the unique site of transthyretin synthesis in the rat eye.

Transthyretin (TTR), or prealbumin, is a 55-kD tetrameric protein which plays an important role in the plasma transport of thyroxine, and through its interaction with retinol-binding protein, of retinol. Four major sources of TTR synthesis have been identified in the mammal: liver hepatocytes, visceral yolk sac endoderm, choroid plexus epithelium, and the eye. We now report in situ hybridization studies demonstrating that in the rat eye, the retinal pigment epithelium is the unique source of TTR synthesis. This finding underscores the developmental, structural, and functional homology between the choroid plexus epithelium and the retinal pigment epithelium. Although the functional significance of ocular TTR synthesis is unclear, it is likely that it serves to transport thyroxine or retinol across the blood-retina barrier, thereby facilitating their effects on differentiation and morphogenesis. Considering the importance of retinol in the biochemistry of the visual process, we propose that TTR may play a role in the intraocular cycling of retinol.

Animals↗
PubMed1990

Distribution of transthyretin in the rat eye.

We reported previously synthesis of transthyretin (TTR), or prealbumin, a transport protein for thyroxine and retinol, in the eyes of rats and cows and showed that in the rat eye, TTR mRNA is localized exclusively in the retinal pigment epithelium (RPE). We now demonstrate by immunohistochemistry that TTR has a more widespread distribution in the rat eye than does its mRNA. Intense immunoreactivity for TTR was found in the RPE, ciliary epithelium, iris epithelium, corneal endothelium, optic nerve fiber layer of the retina, and lens capsule. Depending on the method of processing, immunoreactivity of varying intensity was found also in other ocular structures. In particular, the retinal ganglion cells were strongly immunoreactive on frozen sections but not on paraffin sections. Although vitreous humor was not included in the sections of adult rat eye, sections of a 25-mm rat embryo showed intense immunoreactivity in the vitreous humor. Since plasma TTR does not cross Bruch's membrane into the retina, our findings suggest that ocular TTR is synthesized, at least in part, in the RPE and is transported to specific locations within the eye. Although the physiologic role of ocular TTR is unknown, it is possible that it participates in retinol cycling within the eye. The widespread ocular distribution of TTR may account for the occurrence of various forms of ocular amyloidosis in the familial amyloidotic polyneuropathies, a group of dominantly inherited disorders caused by point mutations in the TTR gene.

Animals↗
PubMed1990

A marker for primary choroid plexus neoplasms.

Primary choroid plexus (CP) tumors are rare neoplasms that present in childhood or, less frequently, in adult life. The majority are benign and amenable to complete surgical excision, but occasionally more invasive variants are encountered. Although generally pathologically distinct, occasionally primary CP neoplasms may be difficult to distinguish from metastatic papillary carcinomas or papillary ependymomas. Conventional cytologic markers are not sufficiently specific to permit accurate diagnosis of primary CP tumors. The authors have reported that the CP is the unique site of synthesis within the brain of transthyretin (TTR, prealbumin), a transport protein for thyroxine and retinol. They therefore investigated the utility of TTR as a biochemical marker for CP tumors. They detected intense immunoreactivity for TTR at high dilutions of primary antiserum in the neoplastic epithelium of all of nine primary CP tumors (six papillomas and three carcinomas), but not in eight cellular or three papillary intracerebral ependymomas, meningiomas, oligodendrogliomas, astrocytomas, primary extracerebral papillary carcinomas (three thyroid, two breast) or five of six cerebral metastases from systemic papillary carcinomas. In one case of cerebral metastasis from papillary thyroid carcinoma, rare isolated immunoreactive cells were observed. Faint staining of the stromal-ependymal junction was seen in myxopapillary ependymomas of the filum terminale, which were otherwise nonreactive. By in situ hybridization, TTR mRNA was abundant in neoplastic CP epithelium, confirming local TTR synthesis. The authors conclude that TTR is synthesized by neoplastic CP epithelium and is an excellent marker for primary CP neoplasms.

Adolescent↗
PubMed1988

Nonidentical distribution of transferrin and ferric iron in human brain.

Using the avidin-biotin immunoperoxidase technique and a diaminobenzidine intensification of the Prussian Blue method, we have compared the distribution of transferrin to that of ferric iron in five normal autopsy brains from adult human males. The observed distribution of transferrin was considerably more widespread than: (1) that of histochemically demonstrable ferric iron; (2) that reported for transferrin in the fetal and neonatal human brain; and (3) that reported for transferrin in other species. Transferrin immunoreactivity was present in neurons, oligodendrocytes, astrocytes, ependymal cells, and choroid plexus epithelial cells, although not in all cells of any type. Ferric iron, on the other hand, was demonstrable only in oligodendrocytes, in myelin sheaths, and possibly in axons. While staining for both transferrin and iron was relatively high in the basal ganglia and substantia nigra, the pattern of staining differed, with striatal efferent fibers staining more heavily than the neuropil for iron and less heavily than the neuropil for transferrin. The choroid plexus, which in the rat has been shown to synthesize transferrin, stained heavily for transferrin and not at all for iron. The findings of low iron and high transferrin in the choroid plexus suggest that the plexus may secrete transferrin into the cerebrospinal fluid, thereby facilitating the translocation of iron within the neuraxis. Furthermore, the nonidentical distribution of ferric iron and transferrin suggests that, in the human brain, transferrin may serve other functions besides the transport of iron from extracellular fluid to cytoplasm.

Adult↗
PubMed1984

Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site.

Nicotinic acetylcholine receptors contain a readily reducible disulfide bond at the periphery of the acetylcholine binding site. Following reduction of this disulfide, the binding site is susceptible to affinity labeling by electrophilic reagents with quaternary ammonium moieties. We reduced purified receptor from Torpedo californica electric tissue and affinity alkylated it with 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The label was incorporated solely into the alpha subunit of the receptor. Isolated, labeled alpha subunit was cleaved with CNBr, and the fragments were separated by reverse-phase high-performance liquid chromatography. A uniquely labeled CNBr fragment was isolated, and its partial sequence was determined by automated Edman degradation. This CNBr fragment was cleaved at tryptophan residues, the subfragments were separated, and the labeled subfragments were partially sequenced. From our protein sequence information, we identify the labeled CNBr fragment as residues 179 to 207 of the sequence of alpha predicted from the cDNA sequence (Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982) Nature (Lond.) 299, 793-797). From the cycle of the Edman degradation in which radioactive residues are released, we conclude that Cys 192 and, possibly in addition, Cys 193 are the residues specifically labeled by 4-(N-maleimido)benzyltri[3H]methylammonium iodide. They are, therefore, close to the acetylcholine binding site.

Affinity Labels↗
PubMedDate not supplied

Extramedullary hematopoiesis within a frontoethmoidal encephalocele in a newborn with holoprosencephaly.

We present the first report of extramedullary hematopoiesis (EMH) in an encephalocele. The patient was a new-born with semilobar holoprosencephaly, a frontoethmoidal encephalocele, and a large subdural hematoma. The encephalocele appeared as a hemorrhagic mass, protruding from the forehead to cover the right eye, without involvement of the sinuses or nasopharynx. Computerized tomography and magnetic resonance imaging studies ruled out other forms of holoprosencephaly and confirmed the continuity of the brain with the extruded mass. Immunohistochemistry confirmed the presence of an atrophic epithelium covering the mass. Histologic examination of the encephalocele revealed EMH both within and adjacent to malformed cerebral cortex, with a tendency for the hematopoietic cells to line up in columns within malformed cerebral cortex. We propose that a single event during the fourth week of gestation could both interrupt closure of the neural tube, giving rise to the encephalocele, and impair migration of the neural crest, leading to holoprosencephaly secondary to failure of neural crest derivatives to induce basomedial telencephalic differentiation. EMH may have been induced from hematopoietic stem cells in the richly vascular meningeal component of the encephalocele, in response to anemia and hypoxia.

Biomarkers↗
PubMedDate not supplied

Myelin staining of archival brain tissue.

To evaluate the feasibility of staining for myelin in archival materials, paraffin blocks were prepared from brain tissue that had been in formalin for intervals ranging from 7 months to over 53 years. Verhoeff and Luxol fast blue stains of the resulting sections yielded staining whose quality was unaffected by duration of fixation. Myelinated and unmyelinated areas were clearly distinguished, and the morphology of individual myelin sheaths was well-preserved. No changes to conventional protocols were required, but it was necessary carefully to monitor the progress of differentiation. With antigen retrieval, it was possible to display immunoreactivity for myelin basic protein. While this persisted even after prolonged fixation, fine detail was lost from the myelin sheaths, and there was staining of oligodendroglial cytoplasm and nuclei, which was not seen in recently fixed tissue. In contrast to this loss of detail in myelin sheaths, immunohistochemistry for glial fibrillary acidic protein displayed astrocytic morphology clearly, even in the oldest tissue. We conclude that archival, formalin-fixed material can be adequately examined for myelin loss and astrocytosis.

Adult↗
PubMedDate not supplied

Archival, formalin-fixed tissue: its use in the study of Alzheimer's type changes.

Preparing for a retrospective study of senile degeneration in schizophrenia, we had occasion to explore the suitability of an old collection formalin-fixed brains and paraffin blocks for study by modern staining methods. Tissue that had been in formalin for 50 years was embedded in paraffin. Sections were then stained with thioflavine S and with immunoperoxidase stains using Alz 50 and antibodies to paired helical filaments, ubiquitin, and beta-amyloid. In all 4 cases that had originally (50 years earlier) received neuropathologic diagnoses of Alzheimer's disease, large numbers of neocortical senile plaques and neurofibrillary tangles were clearly demonstrated by thioflavine S stain and by immunohistochemistry for paired helical filaments, ubiquitin, and beta-amyloid. In each of 4 other cases, in which the original neuropathologic examination had not revealed Alzheimer's disease, no plaques or tangles were observed. Immunoreactivity with Alz 50 was completely absent after 50 years in formalin. Examination of additional cases of Alzheimer's disease revealed that Alz 50 immunoreactivity was well preserved after 10 years in formalin and completely absent after 30 years in formalin. Alzheimer's disease tissue stored in paraffin for 30 years was clearly stained by all modalities. We conclude that immunohistochemical identification of senile plaques and neurofibrillary tangles is practical even after decades of storage in formalin or paraffin. The applicability of techniques that did not exist when these specimens were collected indicates that the systematic, permanent retention of formalin-fixed material may yield unanticipated future benefits.

Adult↗