Lysophosphatidyl choline-induced demyelination. A freeze-fracture study.

Focal demyelination was produced in the rat sciatic nerve by microinjection of lysophosphatidyl choline (LPC). The demyelinating lesion was examined over the following 48 h using the freeze-fracture technique to examine myelin, Schwann cell and axonal membranes. Myelin lamellae were replaced by myriad spherical or oval membranous vesicles. The axonal and Schwann cell plasma membranes remained intact and the latter showed a large increase in caveolae-associated pores in some nerve fibres. The lysis of myelin lamellae and membranous vesicle formation are related to the known action of LPC on myelin and its membrane fusogenic properties. The importance of calcium ion influx and membrane protein aggregation and depletion in vesiculation are discussed.

Distribution of filipin-sterol complexes in the unmyelinated nerve fibre.

The filipin-sterol technique was employed, together with freeze-fracture, to investigate the fine structure of the unmyelinated nerve fibre in the peripheral nerve. No heterogeneity was observed in the distribution of filipin labelling either in the Schwann cell plasma membrane or along the axolemma. The distribution of labelling is contrasted with that in the myelinated nerve fibre and related to the relative morphology and electrophysiology of the two types of fibre.

Filipin-sterol complexes at Schmidt-Lanterman incisures.

Employing the freeze-fracture technique, the distribution of filipin-sterol complexes was determined for membranes of peripheral nerve myelin. A heterogeneous distribution of complexes was observed with the greatest abundance on membranes associated with the cytoplasmic channels of Schmidt-Lanterman and longitudinal incisures. In addition there was an irregular network of well-labelled membrane bands in compact myelin. The results are related to a possible role for these channels and bands in the biochemical turnover of cholesterol in myelin.

Immunocytochemical characterization of 10 pancreatic tumours, associated with the glucagonoma syndrome, using antibodies to separate regions of the pro-glucagon molecule and other neuroendocrine markers.

Histological diagnosis of neuroendocrine tumours can be hampered by their lack of peptide or amine immunoreactivity. In order to assess the usefulness of a range of specific and general markers of neuroendocrine differentiation, 10 pancreatic endocrine tumours, associated with high levels of circulating glucagon, were studied using histology, histochemistry, immunocytochemistry and electron microscopy. All cases showed immunoreactivity for one or other of the peptides derived from pro-glucagon, although only seven were found to contain immunoreactive pancreatic glucagon. The presence of secretory granules in eight of the tumours was demonstrated by electron microscopy, argyrophilia or chromogranin immunoreactivity. Not only was neuron specific enolase positively immunostained in all the tumours, thereby revealing their neuroendocrine nature, but also the intensity of the immunostain was higher in four of the five malignant ones than in the rest of the cases. Pancreatic polypeptide was present in non-glucagon cells in six out of 10 cases. Our results emphasize the importance of the use, not only of general histochemical and immunocytochemical tests but also antibodies to all possible derivatives of the precursor form of the active tumour product in the diagnosis of possible endocrine tumours. In this way, any abnormal molecular forms of the peptide synthesized by tumour cells with altered synthetic and secretory mechanisms may be detected.

Somatostatin-containing D cells exhibit immunoreactivity for rat somatostatin cryptic peptide in six mammalian species. An electron-microscopical study.

Antisera raised against rat somatostatin cryptic peptide (RSCP; corresponding to amino acids 63-77 of rat pro-somatostatin), somatostatin-28-(1-12) and somatostatin-28-(17-28) were used to compare the morphological distribution of these pro-somatostatin-derived sequences within the gastroenteropancreatic system of six mammalian species, including man. Using the immunogold staining procedure, RSCP, SS28-(1-12) and SS28-(17-28) immunoreactivity was found to be present in all the D cells of the tissues investigated. Extra-islet RSCP and SS28-(1-12) immunoreactive cells were also identified in some species. RSCP, SS28-(1-12) and SS-28-(17-28) immunoreactivities were also present in a single case of human duodenal somatostatinoma. Immunostaining of serial ultrathin sections from all specimens in this study revealed that RSCP and both somatostatin immunoreactivities were co-localised in a majority of the reactive cells. Corroborative evidence was obtained by double immunogold staining which further showed that RSCP, SS28-(1-12) and SS28-(17-28) immunoreactivities were co-localised to individual secretory granules in D type cells, both normal and tumour. RSCP and SS28-(17-28) immunoreactivities were invariably co-localised, whereas SS28-(1-12) immunoreactivity was restricted to a sub-population of secretory granules. Our findings suggest that RSCP immunoreactivity is conserved in a number of mammalian species and is stored in each secretory granule type. Consequently, detection of the RSCP sequence may serve as a useful marker for somatostatin-producing systems throughout the diffuse neuroendocrine system.

Filipin-sterol complexes at nodes of Ranvier.

Using the filipin-sterol technique, regional heterogeneity in the axonal and Schwann cell plasma membranes was investigated at the node of Ranvier and paranodes. Filipin-sterol complexes were abundant at the nodal axolemma but infrequent throughout the paranodal axolemma. The paranodal Schwann cell plasma membrane was rich in complexes which extended over the nodal Schwann cell microvilli. There were no regional differences in filipin labelling of the nodal-paranodal Schwann cell plasma membrane in relation to features such as paranodal cytoplasmic columns or mesaxonal furrows. However, the paranodes of adjacent Schwann cells were sometimes markedly different from each other in the amount of filipin labelling. The extent to which filipin labelling is indicative of cholesterol membrane content is discussed and the findings are related to current concepts of distribution, mobility and interaction of protein and lipid in biomembranes, with particular reference to the nodal axolemma.

Medullary carcinoma of the thyroid. An immunocytochemical and histochemical study of 25 cases using eight separate markers.

The current study was undertaken on 25 cases of thyroid medullary carcinoma to compare the diagnostic value of calcitonin with other peptides including PDN-21, the C-terminal flanking peptide of human calcitonin within the calcitonin precursor, and calcitonin gene-related peptide, CGRP. Antiserum raised to chromogranin, an acidic protein of 68,000 daltons, was also used to compare its diagnostic value as a general marker for neuroendocrine neoplasia with neuron-specific enolase (NSE) and Grimelius' argyrophil silver staining. Immunocytochemistry was performed using the peroxidase-antiperoxidase method at the light microscopic level and the immunogold staining procedure at the ultrastructural level. All tumors were reactive to calcitonin and CGRP antisera, whereas PDN-21 was present in 23 cases. It was also found that these peptides were colocalized in the majority of C-cells. The intensity and specificity of CGRP and PDN-21 immunoreaction was comparable to and in some cases even better than that obtained with calcitonin antiserum. In the majority of tumors, somatostatin and bombesin immunoreactivity was either absent, weak, or variable in intensity and distribution. The current study thus demonstrates that together with calcitonin, PDN and, in particular, CGRP antisera may be applied to corroborate immunocytochemical diagnosis in medullary carcinoma of the thyroid. With regard to general neuroendocrine markers, Grimelius' and chromogranin provided the most consistent results. NSE isoenzyme immunoreactivity, on the other hand, was more variable, probably reflecting the metabolic state of the tumor cells.

Localization of glucagon-like peptide (GLP) immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry.

The distribution of peptide immunoreactivities predicted from the sequence of the human preproglucagon gene in enteroglucagon (EG; glicentin-like immunoreactant-containing) cells of the human gut and A cells of the pancreas has been determined by light and electron microscopic immunocytochemistry. At light microscopy the application of peroxidase-antiperoxidase and immunogold-silver staining methods has revealed that glucagon-like peptide (GLP-1 and GLP-2) immunoreactivities coexist with a glicentin-related immunodeterminant in human colorectal EG cells and pancreatic A cells. Using single and double colloidal gold probe electron immunocytochemistry, we have been able to show the coexistence of glicentin, GLP-1, and GLP-2 immunoreactivities within single EG cell secretory granules. No morphologic segregation of the proglucagon immunoreactants was observed in EG cells of the colonic mucosa. In pancreatic A cells we have localized GLP-1, GLP-2, and glucagon-[16-29] immunoreactivities solely to the electron-dense core of the secretory granules, whereas glicentin-related immunoreactivity was restricted to the electron-lucent halo. The results obtained in the present study have shown that the peptide immunoreactivities predicted from cDNA sequencing of the human preproglucagon gene are indeed expressed in colorectal EG and pancreatic A cells. The topographical segregation of immunoreactivities in the A cell secretory granule shows that antigenic determinants derived from the C-terminal portion of proglucagon are stored with glucagon in the core of the secretory granule.

Distribution of filipin-sterol complexes in the myelinated nerve fiber.

Using filipin as a cytochemical probe to reveal the distribution of cholesterol, myelinated peripheral nerve fibers were examined in freeze-fracture replicas. Filipin-sterol complexes were most abundant in the Schwann cell and axonal plasma membranes. In the Schwann cell plasma membrane there was no heterogeneity in complex distribution in relation to the subjacent cytoplasmic network. In myelin lamellae there was a decrease in complexes from outer to inner lamellae and some aggregation of complexes in individual lamellae. The density of complexes in cytoplasmic organelles varied from absent in mitochondria to high in lysosome-like bodies. The results are interpreted in terms of the related biochemical composition and biophysical properties of cell membranes, with particular reference to the myelinated nerve fiber. The influence of diffusion barriers and gradients on the formation of complexes by filipin is considered.

Localization of Tamm-Horsfall-glycoprotein-like immunoreactivity in cultured baby-hamster kidney cells, shown by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques.

Tamm-Horsfall glycoprotein was isolated from hamster urine, and antiserum against it was produced in rabbits. IgG was isolated from the antiserum. Immunocytochemical methods were used to localize Tamm-Horsfall-like immunoreactivity in three substrains of baby-hamster kidney (BHK) cells. Indirect immunofluorescence techniques showed that, in two substrains (BHK-21/C13/2P and BHK-21/C13/3P), a proportion of the cells fluoresced brilliantly, whereas those of the third substrain (BHK-21/ICRF) were totally negative. Related findings were obtained by the immunoperoxidase optical-microscopic technique. From the results of immunoperoxidase techniques using the electron microscope, it was concluded that the substance was present in association with the plasma membranes of the reacting cells. Our data suggest that the line of baby-hamster kidney cells, BHK-21/C13, may contain cells of renal-tubular epithelial origin, and that the proportion of these may be variable from one subculture to another.

Visualisation of messenger RNA directing peptide synthesis by in situ hybridisation using a novel single-stranded cDNA probe. Potential for the investigation of gene expression and endocrine cell activity.

The neuropeptide tyrosine precursor (pre-pro-NPY) messenger RNA (mRNA) has been localised in formaldehyde-fixed human phaeochromocytoma tissue using a sensitive in situ hybridisation procedure and a novel single-stranded cDNA probe. The reaction product was revealed by avidin-biotin-peroxidase complex and streptavidin-gold complex with silver enhancement. This technique may be applied for the determination of biosynthetic activity of endocrine and neuronal cell bodies. This is largely due to its rapidity by comparison with conventional autoradiographic procedures, to the permanence of the reaction product and to the sensitivity of the visualisation steps.

Localization of Tamm-Horsfall glycoprotein in the normal rat kidney and the effect of adrenalectomy on its localization in the hamster and rat kidney.

Immunofluorescence and immunoperoxidase techniques were used to study the localization of Tamm-Horsfall glycoprotein in the normal rat kidney. In the fluorescence microscopical preparations, the glycoprotein was observed in the thick ascending limbs of the loops of Henle and distal convoluted tubules and was thus, in general, similar to our earlier observations on the hamster and man. The situation in the maculae densae was, however, somewhat different, for in the rat the majority of them were seen to possess Tamm-Horsfall glycoprotein on the luminal surfaces of their cells and only a small proportion resembled the hamster and man in lacking it. These observations were confirmed by the immuno-electron microscope technique. Furthermore, it was shown that in the thick ascending limbs and distal convoluted tubules, Tamm-Horsfall glycoprotein is associated with the total plasma membrane systems of its cells. Thus it appears that Tamm-Horsfall glycoprotein is confined to that part of the nephron responsible for the process of urine dilution. As this function is, at least in part, regulated by adrenal cortical hormones, the effect of adrenalectomy on the distribution of the glycoprotein was studied. The results obtained showed varying degrees of disappearance of Tamm-Horsfall glycoprotein in the kidneys of adrenalectomized hamsters, initially from the distal convoluted tubules and later from the thick ascending limbs. In the rat, on the other hand, the effect of adrenalectomy on Tamm-Horsfall glycoprotein was much less pronounced, possibly due to the presence of secondary adrenal tissue. The possible physiological significance of these findings is discussed.

Substance P-like immunoreactivity in the intramural nerve plexuses of the guinea-pig ureter: a light and electron microscopical study.

Immunofluorescence and immunoperoxidase techniques were used to study the distribution of substance P-like immunoreactivity in the intramural nerve plexuses of the guinea-pig ureter. In light microscopical preparations, immunoreactivity was observed in plexuses related to the muscle coat as well as in plexuses in the submucosa and beneath the epithelium. Ultrastructural examination showed that the immunoreactivity was located primarily in axons in the nerves. In perfusion-fixed specimens, there was evidence of its presence both in axons with terminals containing mainly large dense-cored vesicles and in axons with terminals containing mainly small vesicles. The presence of substance P-like immunoreactivity in axons with terminals containing mainly large dense-cored vesicles was supported by examination of specimens treated in vitro with capsaicin. In these specimens, the axons were dilated and showed a number of other changes in fine structure. There was also a substantial reduction in the amount of immunoreactivity in the nerve plexuses and the dilated axons contained little if any reaction product. The possibility that axons which contained large amounts of reaction product after treatment with capsaicin represented axons with terminals containing mainly small vesicles was discussed. Comparison of the distribution of the different types of small vesicle-containing terminal identified in glutaraldehyde-fixed material with that of axons containing reaction product suggested that the immunoreactivity present in such axons was located in those in which the small vesicles in the terminals were clear rather than those in which the vesicles contained dense material.

Localization of Tamm-Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques.

Small pieces of tissue obtained from apparently normal areas of four surgically removed adult human kidneys were used in the present study. The results obtained by immuno-fluorescence and immuno-electron microscopical techniques show that Tamm-Horsfall glycoprotein (THP) is present in the thick ascending limbs of the loops of Henle and the distal convoluted tubules. Within the cells concerned, the protein is associated with the luminal, lateral as well as basal, plasma membranes and their infoldings. The cells of the macula densa are completely negative as are those of proximal convoluted tubules, glomeruli and collecting ducts. The possible significance of these findings in relation to the process of urine dilution in the nephron is discussed.

Light and electron microscopical observations on the macula densa of the Syrian hamster kidney.

The ultrastructure of the macula densa of the hamster kidney is, in general, similar to that reported for the few other species which have been studied. Their structure indicates that macula densa cells differ in a number of important respects from those of the rest of the thick ascending limb and the distal convoluted tubule. Among these differences may be mentioned the more widely distributed mitochondria with their circular and elliptical profiles; the extensive subsurface vacuolation and the irregular and shallow folds of the basal plasma membrane. Unlike some reports on other species, however, the Golgi complexes were not restricted to an infranuclear position, but were observed in a variety of situations. It was also observed that T-H glycoprotein characteristically associated with the plasma membranes of the cells of the thick ascending limb and the distal convoluted tubule was invariably absent in the macula densa. The possible physiological implications of this and the other observations are discussed.

Localization of Tamm-Horsfall glycoprotein in the fetal and neonatal hamster kidney as demonstrated by immunofluorescence and immunoelectron microscopical techniques.

Tamm-Horsfall (T-H) glycoprotein was demonstrated in the developing hamster kidney using immunofluorescence and immunoelectron microscopical techniques. The glycoprotein was first observed in the fetal kidneys on the 12th day of gestation and was confined to the luminal surface of the presumed distal tubules of the medulla. It was not until the 14th day of gestation that T-H glycoprotein was also sometimes seen to be associated with the lateral and basal invaginations of the plasma membranes of the now differentiated distal tubules. On the 16th day (1st day post-partum) the glycoprotein was also found in the cortex. Although the general distribution of T-H glycoprotein was at 3-4 days after birth similar to the adult, the full intensity of staining was not attained until after the 21st day. The possible physiological significance of these findings is discussed.

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm-Horsfall glycoprotein in adult hamster kidney.

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.