Electron Microscopic Demonstration Research Articles

Neurologic involvement in glucagonoma syndrome.Response to combination chemotherapy with 5-fluorouracil and streptozotocin

A 34-year-old man presented with classic glucagonoma syndrome manifested by weight loss, dermatitis, stomatitis, anemia, and mild diabetes mellitus. The diagnosis of glucagonoma was made by light and electron microscopic demonstration of a metastatic alpha cell carcinoma in a liver biopsy specimen. Plasma glucagon concentration was abnormally high. The patient also had symptoms and signs of involvement of the central nervous system. Radionuclide and CAT scans of the brain, negative CSF cytology and myelography excluded the possibility of metastases or other space-occupying lesions. Glucagon was demonstrated in the CSF. We postulate that the neurologic symptoms were due to direct or indirect effect of this hormone on the brain. Following therapy with streptozotocin and 5-fluorouracil, the patient had a subjective and objective clinical and hormonal remission of his disease including amelioration of his neurological impairment.

Peripheral intraaxonal storage in Tay-Sachs' disease (G M2-gangliosidosis type 1

Considerable intraaxonal lipid storage was observed in intramuscular nerve fibres mainly of the extraocular muscles and less frequently in limb muscles of a 3-year-old child, which had suffered from Tay-Sachs' disease. Many axons of the small intramuscular nerve twigs and of the terminal and preterminal nerve endings showed spherical and cylindrical enlargements containing granular storage material of the same staining properties as the material in the central nervous system. The identity of the axonal and the neuronal storage material was further confirmed by electron-microscopical demonstration of typical multilamellated cytoplasmic bodies (MCBs) in both of them. In addition, silver impregnation and electron microscopy revealed dystrophic changes with an increase of argyrophilic filamentous material within some of the axonal distensions, indicating that the latter were probably due to both a lipid storage and an unspecific dystrophic process.

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

Application of coupled oxidation reaction to electron microscopic demonstration of horseradish peroxidase: cobalt-glucose oxidase method

PH gradient electrophoresis and isoelectric focusing of lipoproteins on agarose bead thin layers.

A new method of isoelectric focusing (IEF) and pH gradient electrophoresis, using thin layers of agarose gel beads, was devised to investigate chylomicrons and very low density lipoproteins (VLDL). pH gradient stability and cathodal gradient drift were similar to those of polyacrylamide gel IEF, and linearity of gradients was maintained for 23 hr. Chylomicrons and VLDL were detectable without staining. Chylomicrons from human serum and from rat lymph migrated in this system. Rat lymph chylomicrons, obtained by ultracentrifugation, migrated in several discrete bands, and this heterogeneity or rat chylomicrons was confirmed by electron microscopic demonstration of chylomicrons in each band. This new technique has permitted the first measurement of isoelectric points of some lipoproteins in the ultracentrifuged fraction of human serum chylomicrons and the first separation of multiple discrete fractions of ultracentrifuged lymph chylomicrons.

Sustained growth and three-dimensional organization of primary mammary tumor epithelial cells embedded in collagen gels.

We have developed a method for embedding cells within a collagen matrix which allows sustained growth of mouse mammary tumor epithelial cells in primary culture. A characteristic and reproducible pattern of organization and growth occurs: the cells rearrange themselves and produce duct-like structures extending into the matrix, resulting in a three-dimensional outgrowth. Autoradiography showed continuous [3H]thymidine incorporation during 8 weeks in culture. An increase in DNA content of the cultured cells as a function of time was observed. Mouse mammary tumor cells cultured in the conventional monolayer system failed to show any significant increase in cell number during a culture period of 6 weeks. In addition, in such monolayer systems, cells progressively became detached from the dishes in long-term culture. The mammary epithelial cell origin of the collagen gel cell outgrowths was shown by electron microscopic demonstration of polarized cells containing tight junctions and budding mammary tumor virus particles. In addition, in vivo transplantation of collagen gel outgrowths resulted in the development of mammary adenocarcinoma histologically similar to the donor tumor. Cellular outgrowth patterns resembling those from tumor cells were also seen in similar collagen gel cultures of normal mammary cells from mouse and human and of hyperplastic alveolar nodule cells from mouse. The significance and usefulness of this system in comparison to the conventional monolayer system are discussed.

Electron microscopic demonstration of centrifugal nerve fibers in the human optic nerve

Electron microscopic views of centrifugal nerve fibers in the optic nerve stump of a 56-year-old man are presented. These nerve fibers had survived for 16 days after removal of the corresponding eyeball and exhibited terminal swellings pointing in a distal direction and indicating axoplasmic flow towards the removed eye. The centrifugal nerves in this adult lack any evidence of attempted regeneration that has earlier been observed under similar conditions in the optic nerve stump of a child.

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes

The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope.

Human ecthyma contagiosum (ORF)

A case of human ecthyma contagiosum (orf), diagnosed histologically, is described. The diagnosis was confirmed by electron microscopic demonstration of orf virus in desquamated epidermal cells. Clinical and histological aspects of the disease are discussed.

Electron microscopic demonstration of a supraependymal cluster of neuronal cells and processes in the hamster third ventricle.

A supraependymal cluster of neuronal cells and processes consistently present on the floor of the hamster third ventricle was identified and characterized by means of correlative scanning (SEM) and transmission (TEM) electron microscopy. SEM revealed each cluster to be ovoid with the majority of its surface covered by dome-shaped protrusions and fine beaded fibers. A number of processes traveling individually or in groups also entered or exited from the cluster at its base. As these processes passed over the ventricular surface, they contributed to an extensive network on the floor and ventral aspect of the ventricular wall. Some processes terminated on the ependymal surface in bulbous endings while others penetrated the ependyma. The neuronal nature of these clusters and their associated processes was confirmed at the TEM level. The dome-shaped protrusions visible on the surface of the cluster in SEM corresponded to apical surfaces of neurons confined to the peripheral aspect of a core of loosely arranged processes. These cells exhibited a prominent nucleolus, stacks of rough endoplasmic reticulum (RER), polyribosomes, Golgi cisternae, mitochondria and microtubules (MT) and gave rise to dendritic processes which extended into the core. These dendrites gave off branches at acute angles and contained polyribosomes, single cisternae of RER and evenly spaced MT. Other profiles of processes within the core shared these characteristics, suggesting that they also were branches of the peripheral cells. Axons present within the core and on the cluster's surface exhibited vesicle-filled varicosities which frequently established synaptic contact with the peripheral cells and their processes. The presence of an intraventricular cluster of neurons which potentially communicates with centers extrinsic to the ventricle may have important implications in the hypothesized role of cerebrospinal fluid and tanycytic ependyma in the neuroendocrine regulation of anterior pituitary function.

Electron-microscopic demonstration of terminal and internal initiation sites for cDNA synthesis on vitellogenin mRNA.

cDNA synthesized on purified vitellogenin mRNA from Xenopus liver was hybridized to the template in formamide/urea at 22 degrees C to avoid degradation of the RNA. The hybrids formed were visualized by spreading for electron microscopy. Contour length measurements proved that most of the RNA molecules in the hybrids were still intact showing the expected molecular weight of 2.3 x 10(6). The hybridized cDNA corresponded on the average to 12% of the RNA length. In about 80% of the molecules the cDNA was located at one end. Since cDNA synthesis was primed by oligo(dT), the terminal duplex region marks the 3' end of the vitellogenin mRNA molecule. Internal duplex regions were mainly located at a specific position starting about 2800 nucleotides from the 3' end. Since the cDNA hybridizing at the internal position could specifically be synthesized on a vitellogenin RNA fragment isolated on poly(U)-Sepharose as an oligo(A)-containing RNA, we conclude that cDNA synthesis is not only initiated by the poly(A) of the 3' end, but also by a specific internal sequence.

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and non-denaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosomic DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50-75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multi-nucleosomic chromatin segments containing a full complement of histones.

Lymphocyte surface immunoglobulin in Xenopus laevis light and electron microscopic demonstration by immunoperoxidase method

To study the distribution of surface immunoglobulin (sIg * ), splenic and thymic lymphocytes of Xenopus laevis were exposed to anti— Xenopus Immunoglobulin rabbit IgG conjugated with horseradish peroxidase (aXIg-HRP). Light microscopy of lymphocytes from 6 – 8 months old toadlets revealed that sIg was detectable on 53% splenic and 91% thymic cells. “Cap” formation concomitant with internalization of sIg upon warming was evident in both splenocytes and thymocytes. The sIg positive cells in spleen were mostly small lymphocytes, although some were plasma cells, medium- and large lymphocytes. In spleen of toadlets thymectomized at Nieuwkoop and Faber stage 45 (4 days postfertilization), more than 90% of lymphocytes expressed sIg. For the spleen, lymphocytes bearing sIg seem to be thymus-independent. * Abbreviations used in this paper: aXIg, anti- Xenopus immunoglobulin rabbit IgG; aXIg-HRP, aXIg conjugated with horseradish peroxidase; HRP; horseradish peroxidase; sIg, surface immunoglobulin.

Inclusion body myositis

We report six cases of inclusion body myositis (IBM), a distinct but infrequently recognized inflammatory disease of skeletal muscle. Clinically, IBM differs from dermatomyositis and polymyositis. It lacks features of collagen-vascular disease, has a relatively benign and protracted course, frequently involves distal muscles, is found mainly in males, and does not improve with corticosteroid treatment. Electronmicroscopic demonstration of abnormal filaments in muscle cells is necessary for definite diagnosis, but IBM may be suspected by the finding on cryostat sections of numerous hematoxylinophilic granules in "lined" vacuoles in muscle cells. These correspond to whorls of cytomembranes. Although in dermatomyositis the capillary network is partly destroyed, in IBM it is usually augmented. A viral etiology of IBM has been suggested but remains unproven.

Synapses between identified neuronal elements. An electron microscopic demonstration of degenerating axon terminals synapsing with Golgi impregnated neurons

Golgi impregnated neurons can be gold-toned, so that in the electron microscope their profiles are marked by gold particles. It is shown that this technique can be used to determine which neurons in the cerebral cortex synapse with degenerating axon terminals. Thus, both the pre- and post-synaptic components of thalamocortical synapses can be identified.

Mammalian pancreas development: Regeneration and differentiation in vitro

Studies of the temporal sequence of mammalian pancreas development have demonstrated the existence of a protodifferentiated state that is characterized by low constant levels of amylase specific activity. In order to investigate this state further, guts from 9-day mouse embryos were cultured for 3 days, during which a protodifferentiated dorsal pancreas formed, consisting of a neck region or duct that extended from the intestinal wall and terminated in a large bulb of tissue. The pancreas bulb was then removed, leaving only the duct. This day corresponded to Day 12 of gestation. During 5 days of additional culture, 37 of 43 ducts regenerated a pancreas with an amylase specific activity equal to that of control pancreas (2.4 μg of maltose hydrate released/min/μg of protein at 37°C). Guts placed in culture at 12 days, with removal of the pancreas the same day, gave similar results. Other combinations of starting embryonic age plus culture time, prior to pancreas removal, totaling 12 or 13 days also underwent regeneration. Only of 18 pancreases regenerated from ducts equivalent to 14 days of gestation. Removal of the stomach, the intestine, and the ventral pancreas did not affect the ability of the dorsal pancreas to regenerate. However, removal of the duct at the presumptive intestinal wall did not result in regeneration. Regenerated pancreas differentiation was further confirmed by electron microscopic demonstration of zymogen granules in exocrine cells and of at least two types of secretory granules in endocrine cells. The results demonstrate that the protodifferentiated duct can regenerate a new pancreas including both exocrine and endocrine tissue, and that the regenerated pancreas is not retarded in its development when compared with the control pancreas.

Primary carcinoma of the trachea

Two cases of primary carcinoma of the trachea are described. Both patients were female and had smoked cigarettes for many years. One of the tumours was a squamous cell carcinoma and the other an oat cell carcinoma, the commonest and the rarest forms of tracheal cancer respectively. The diagnosis in the case of oat cell carcinoma was confirmed by electron-microscopic demonstration of neurosecretory granules, a unique observation at this site. Oat cell carcinoma of the trachea does not appear to have been recorded previously in a female.

Electron microscopic demonstration of glycogen and phosphorylase activity in rat hepatocytes.

The effect of fixation with a bicarbonate-buffered solution of paraformaldehyde and polyvinyl pyrrolidone (PVP) on the ultrastructural demonstration of glycogen and phosphorylase activity in rat hepatocytes has been studied. Phosphorylase was demonstrated by the precipitation of liberated phosphate ions with ferrous ions. 7.5% PVP was included in all steps in the procedure before post-fixaiton in osmium tetroxide. Glycogen particles were well preserved. Structures connecting membranes and glycogen particles were also evident. Phosphorylase activity was rapidly inhibited by the fixative; the fixation time was, therefore, kept very short. The final reaction product was localized on glycogen particles and on endoplasmic membranes in association with glycogen particles. The results support the view that endoplasmic membranes are involved in the metabolism of glycogen in hepatocytes.

Electron microscopic immunocytochemical demonstration of separate mesotocinergic and vasotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

With the use of the unlabeled antibody peroxidase-antiperoxidase (PAP) method at the electron microscopic level, it has been shown that the pars intermedia of the hypophysis of Rana temporaria contains a diffuse intercellular network of separate mesotocinergic and vasotocinergic nerve fibres.

赤血球膜燐脂質に関する電顕組織化学的研究

赤血球膜燐脂質に関する電顕組織化学的研究

Electron microscopic cytochemical demonstration of adenyl cyclase activity in psoriatic epidermis.

Isoproterenol and sodium fluoride stimulated adenyl cyclase activity was detected in epidermal tissue from 2 patients with untreated psoriasis by an electron microscopic cytochemical technique. Adenyl cyclase activity was present on the outer surface of the cell membranes, predominantly in the basal cells and in the 4-5 lower Malpighian cell layers, while the superficial layers, stratum granulosum and stratum corneum showed no activity. The precipitates (lead-PPi complexes) after isoproterenol stimulation were larger and fewer in number than those seen after sodium fluoride stimulation. Isoproterenol stimulation was abolished by propranolol. Neither the uninvolved epidermis from the 2 patients with psoriasis nor the normal skin from 2 volunteer individuals showed any difference from the psoriatic epidermis.