Secretory pathways and processing of human proopiomelanocortin (POMC) using transformed mouse cultured fibroblasts (L cells) and AtT20 cells by human POMC gene--preembedding immunoelectron microscopic studies.

In order to elucidate the relationship between secretory pathway and processing for precursor molecule of peptide hormones, we performed immunoelectron microscopic studies to localize POMC-derived peptides in mouse cultured L cells (fibroblasts without secretory granules) and in mouse AtT20 cells (ACTH secreting pituitary tumor cells with secretory granules) which had been transformed with human POMC gene. From the electron microscopic localization patterns, L42 cells were considered to serve as a model of constitutive pathway without processing of POMC, and A53 cells were considered to serve as a model of transgranular (regulated) pathway with processing of POMC. Immunoblotting supported these interpretations.     

Morphological studies on neuroglia

Summary The silver-impregnation procedure of Tsujiyama is suitable for demonstration of all three classical types of neuroglial cells; in the present study it was used for electron microscopic identification of neuroglial cells in the brain of the cat. The aim of the present study was 1) to determine impregnated structural correlates of neuroglial cells at the light- and electron-microscopic levels, and 2) to determine whether the method of Tsujiyama is applicable for the electron microscopic identification of the single types of neuroglial cells. Silver deposits were observed over the cytoplasm and processes of astrocytes where numerous glial filaments were present. Oligodendrocytes and microglial cells may be precisely differentiated by use of Tsujiyama's silver impregnation method at the electron microscopic level due to the pattern of silver-deposition in these two basic types of cells. This silver-impregnation method combined with electron microscopy is thus suitable for a precise identification of neuroglial cells; the technique may prove to be very helpful in identification of such categories of neuroglial cells that encompass also the images of cells which cannot be classified by use of the standard methods.Supported by a grant (No. 437002) from the Ministry of Education, Science and Culture, Japan     

Staining patterns for the anti-horseradish peroxidase antibody reaction in proplasma cells developing in the medulla of rat popliteal lymph nodes during the secondary response

The development of plasma cells from lymphocytes was studied in the medulla of popliteal lymph nodes of rats during the secondary response to horseradish peroxidase (HRP). Changes in the microscopic appearance of proplasma cells were compared with changes in the intensity of the anti-HRP antibody reaction in these cells. Early proplasma cells, appearing 2 to 3 days after the injection of HRP into the footpads, were relatively small cells similar in size to lymphocytes. Their small nuclei were eccentrically located due to the one-sided enlargement of the pyroninophilic cytoplasm. The reaction for the anti HRP antibody in these cells was weak or negative. Other proplasma cells located in the same medullary cord regions showed a more intense antibody reaction. This change was correlated, in many cases, with an enlargement of the nucleus, giving the cells a blast-like appearance. Three to 6 days after the reinjection of the antigen, the medullary cords contained many mature plasma cells characterized by an intense antibody reaction. The mature plasma cells were always accompanied by proplasma cells, the latter varying in microscopic appearance (stage of development) asd staining intensities (antibody contents). The staining intensities and the microscopic appearance of proplasma cells, and the proportion of proplasma cells to plasma cells, varied in different medullary cord regions of the same lymph nodes. The staining patterns, together with the microscopic appearance of the cells, seemed to show whether antibody formation was inhibited or stimulated.     

Electrical impedance assays of blood cells

In this review, the capability of electrical impedance spectroscopy analysis of blood cells, especially for red blood cells is presented, highlighting its large area of related biomedical relevance. The method is briefly introduced and basic theoretical aspects are discussed by considering both phenomenological (e.g. equivalent circuit) and microscopic approaches. The latter include a comparative analysis of the relevance of considering real shape (consistent with microscopic observations) versus spheroidal approximations (prolate and oblate spheroids) with the same surface and volume concentration. We show that while ellipsoidal approximation is fairly good for randomly oriented cells, it is quite poor whenever oriented cells are measured. The voluminous literature on the electrical analysis of blood cells is reviewed to stress the most promising biomedical applications of the method either per se or in combination with complementary e.g. (micro) fluidic approaches.     

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.     

Immunoelectron microscopic localization of epidermal growth factor in the eccrine and apocrine sweat glands.

We studied the localization of the epidermal growth factor (EGF) in eccrine and apocrine sweat glands with light microscopic and electron microscopic immunohistochemistry. Anti-human EGF (anti-hEGF) polyclonal antiserum and anti-hEGF monoclonal antibody (MAb) were used for the study. Light microscopic immunohistochemistry with monoclonal and polyclonal antibodies showed that hEGF-like immunoreactivity was strongly positive in the myoepithelial cells and weakly positive in the secretory cells of eccrine sweat glands. In apocrine sweat glands, it was strongly positive in the secretory cells as well as in the myoepithelial cells. Immunoelectron microscopy with polyclonal antibody showed that hEGF-like immunoreactivity was present in secretory granules of apocrine secretory cells. These granules had mitochondrion-like internal structure. No reactivity was observed on the eccrine secretory cells by immunoelectron microscopy. Neither dark cell granules nor mitochondria in eccrine secretory cells were labeled with anti-hEGF antibody. In both eccrine and apocrine sweat glands, hEGF-like immunoreactivity was diffusely present in the cytoplasm of myoepithelial cells. However, nuclei and mitochondria of myoepithelial cells were devoid of immunoreactivity for hEGF. Our observations indicate that apocrine sweat glands may secrete more hEGF in the sweat than eccrine sweat glands.     

Use of the indirect immunofluorescence method for detection and enumeration of Escherichia coli in seawater samples

The determination of Escherichia coli in marine waters through a rapid method, the microscopic indirect immunofluorescent technique, is evaluated in comparison with the conventional count on m-FC agar medium. The data obtained in seawater samples, collected monthly along the Messina coastline, show good sensitivity of the analysis and agreement between the microscopic and culture technique, with a detection limit of 10(2) cells 100 ml(-1) for immunofluorescence.     

A tentative direct microscopic method for counting living marine bacteria.

Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.     

Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.     

Ascitic fluid cytologic features of a malignant mixed mesodermal tumor of the ovary

A case of malignant mixed mesodermal tumor of the ovary in a postmenopausal patient presenting with abdominal distension is reported. Cytologic examination of smears of the ascitic fluid showed the presence of adenocarcinomatous and sarcomatous cells (with some of the latter being giant cells) plus numerous unidentifiable cells that bore some resemblance to either mesothelial cells or macrophages. Electron microscopic studies showed a clear differentiation of the adenocarcinomatous and sarcomatous cells from positively identified mesothelial cells and macrophages also present in the ascitic specimen, indicating that the unidentified cells in fact originated in the adenocarcinoma (endometrioid carcinoma), chondrosarcoma and unclassified sarcoma found in the surgical specimen. The differential diagnostic cytomorphologic and electron microscopic features are described in detail.     

Probing the behaviors of gold nanorods in metastatic breast cancer cells based on UV-vis-NIR absorption spectroscopy

In this work, behaviors of positively-charged AuNRs in a highly metastatic tumor cell line MDA-MB-231 are examined based on UV-vis-NIR absorption spectroscopy in combination with inductively coupled plasma mass spectrometry (ICP-MS), transmission electron microscopy (TEM) and dark-field microscopic observation. It is found that characteristic surface plasmon resonance (SPR) peaks of AuNRs can be detected using spectroscopic method within living cells that have taken up AuNRs. The peak area of transverse SPR band is shown to be proportionally related to the amount of AuNRs in the cells determined with ICP-MS, which suggests a facile and real time quantification method for AuNRs in living cells. The shape of longitudinal SPR band in UV-vis-NIR spectrum reflects the aggregation state of AuNRs in the cells during the incubation period, which is proved by TEM and microscopic observations. Experimental results reveal that AuNRs are internalized by the cells rapidly; the accumulation, distribution and aggregation of AuNRs in the cells compartments are time and dose dependent. The established spectroscopic analysis method can not only monitor the behaviors of AuNRs in living cells but may also be helpful in choosing the optimum laser stimulation wavelength for anti-tumor thermotherapy.     

Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations

A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.     

Enhanced adhesion/spreading and proliferation of mammalian cells on electropolymerized porphyrin film for biosensing applications

A polymer film of porphyrin was formed through electropolymerization of p-amino-substituted tetraphenylporphyrin on indium tin oxide (ITO) surfaces. The adhesion and proliferation of MCF-7 cells (human breast cancer cell line) on the film were investigated. It was found that cells cultured on this film could attach and spread more rapidly than on glass, ITO and tissue culture polystyrene (TCPS), and thus the film was demonstrated to be a good adhering substrate. MTT experimental results show that the viability of cells cultured on this film is higher than on TCPS, and fluorescence microscopic observation indicates that cells cultured on the film are not under apoptosis. Based on its excellent cytocompatibility, the polyporphyrin film was used to modify the gold electrode surface of a piezoelectric quartz crystal, and quartz crystal microbalance (QCM) technique was applied for real-time monitoring of MCF-7 cell growth and assessment of chemical cytotoxicity. The proliferation and condition of cells on the surface of the film-modified quartz crystal gold electrode were investigated through fluorescence microscopic observation. The results obtained from QCM experiments are consistent with that from microscopic observation. Additionally, the polymerized film on gold surface can be removed completely and easily, which greatly improves the reproducibility of the quartz crystal gold electrode.     

Differentiation of human T and B peripheral blood lymphocytes by high-resolution cell image analysis

Automated cell image analysis of light and electron microscopic pictures was used for differentiation of nonlabeled lymphocytes in blood smears and in smears of purified lymphocyte suspensions. The percentages of T and B lymphocytes were determined by a two-step rosette assay with sheep red blood cells (T cells) and an immunofluorescence assay with FITC-labeled antihuman globulin (B cells). Images from 1,400 Feulgen-stained and 12,000 Pappenheim-stained cells were analyzed. Various classification methods allowed two lymphocyte subpopulations to be discriminated at the light and electron microscopic levels on the basis of different visual and subvisual morphologic features. As found by immunologic methods, morphologically determined subpopulations corresponded to T and non-T cells, with no further differentiation of non-T cells into B or null cells possible. The results allow the conclusion that there are morphologic differences between human T and non-T cells, with the differences distinguishable from individual variations as well as from alterations induced by sample preparation.     

Investigation of staining,polarization and fluorescence microscopic properties of myoepithelial cells

Summary During studies of early arteriosclerotic lesions fibers with the staining properties of myosins were observed in epithelial cells of various organs. To obtain a basis for further studies, staining, oolarization and fluorescence microscopic properties of classical myoepithelial cells and tonofibrils were investigated. The tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN stain for myosins and related proteins was applied to sections of tongue and skin. In other series various milling dyes, xanthene dyes and Thiazine Red R were substituted for Levanol Fast Cyanine 5RN.Myoepithelial cells of lingual and eccrine sweat glands showed the microscopic properties of smooth muscle cells; tonofibrils had little or no affinity for the dyes tested. The terminal bar-terminal web system of glandular epithelium and the fibrous layer in ducts of eccrine sweat glands resembled myosins and differed significantly from proteins of the epiderminkeratin group, e.g. tonofibrils. In preliminary studies the iodinated xanthene dyes Rose Bengal G, Erythrosin B and Y were found suitable for light, fluorescence and electron microscopic studies.     

Immunocytochemical localization of monoamine oxidase type B in enterochromaffin-like cells of rat oxyntic mucosa

We used immunohistochemistry to identify the localization of monoamine oxidase type B (MAOB) in the rat oxyntic mucosa. At light microscopic levels, MAOB-immunopositive cells were mostly located in the basal half of the oxyntic mucosa. By a double-labeling immunofluorescence method, it was shown that MAOB immunoreactivity was localized in almost all of histidine decarboxylase (HDC)-positive cells. Only a few MAOB-positive cells were negative for HDC. At electron microscopic levels, immunohistochemical reaction products of MAOB were detected on the mitochondrial outer membranes in cells that showed morphological characteristics of enterochromaffin-like (ECL) cells. These findings indicate that ECL cells contain MAOB in the rat. We provide a hypothesis that MAOB is involved in the inactivation mechanism of histamine that is released from ECL cells and activates parietal cells to secrete gastric acid.     

Autoradiographic study of binding and internalization of follicle-stimulating hormone in the mouse testis minces in vitro

The internalization of FSH-receptor complexes was demonstrated in mouse testis by means of light and electron microscopic autoradiography. Chopped testicular pieces were incubated with radioiodinated FSH (131I-NIADDK-rat FSH-I-4) for 10, 20, 60 and 180 min. After incubation the pieces were fixed with glutaraldehyde containing tannic acid, and embedded in Spurr's resin. Semithin and ultrathin sections were cut for light and electron microscopic autoradiography, respectively. In light microscopic autoradiographs, silver grains were preferentially localized over Sertoli cells, regardless of incubation time. Sixty to 70% of the total number of grains were located over Sertoli cells which account for only about 4% of the total cell population of the seminiferous tubules. The majority of these grains correspond to the specific FSH binding sites, because few grains remained after incubation with an excess amount of unlabeled FSH. In electron microscopic autoradiographs, the half-distance (HD) value for the 131I-labeled line source was about 216 nm in the present study. After 10 min of incubation, 56.6% of the total number of silver grains were located over the plasma membrane of Sertoli cells. In testicular pieces incubated for longer periods (20, 60 and 180 min), both the percentage and relative concentration of grains increased in the Golgi apparatus and lysosomes and decreased in the plasma membrane. These results suggest that [131I]iodo-FSH first binds to FSH receptors on the plasma membrane of Sertoli cells, then FSH-receptor complexes are internalized. The increase in the number of grains over the lysosomes following longer incubation, indicates that internalized [131I]iodo-FSH or FSH-receptor complexes are subjected to degradation.     

Morphology, differentiation and matrix production of liver cells in organoid cultures (high density cultures) of fetal rat livers.

The aim of this study was to demonstrate the morphology and matrix synthesis of embryonic rat liver cells (day 18 of gestation) in organoid cultures (high density cultures) with electron microscopic and immunomorphological techniques. For this purpose the cells of embryonic rat livers were isolated enzymatically and grown in an organoid culture (high density culture) for 3 weeks in a Trowell system. During the first 48 h a sorting-out process took place, i.e. liver and blood-forming cells met to form aggregates. In between mesenchymal cells were seen. Vessel-like cavities developed. Electron microscopic inspection of the hepatocytes did not reveal any lesions of the cell organelles after 14 days in culture. As late as after a 3-week culture period mitochondrial swellings and an increased number of autophagic vacuoles were observed. A rim of collagenous fibrils or fibrillar bundles and granular matrix structures was perceptible as early as after 7 days in culture. Immunofluorescence microscopic techniques revealed collagen types III, IV and VI as well as laminin, nidogen, heparansulfate-proteoglycan and fibronectin in these areas. Thus, the composition of the matrix in this culture system corresponds (apart from the absence of collagen type I) to the embryonic situation. Therefore, the organoid culture appears to be an appropriate technique to study the behaviour of hepatocytes in vitro. It is especially suited to demonstrate the formation of matrix components in liver cells and their extracellular occurrence.     

Electron microscopic and ultracytochemical investigations of thyroid C cells in primary hyperparathyroidism

The subject of study were C Cells of the human thyroid in course of primary hyperparathyroidism caused by the presence of a single adenoma. C cells were estimated by routine electron microscopic technique and by cytochemical cholinesterase (ChE) reaction according to Iwayama in electron microscopic technique. The presence of single C cells and of a small agglomeration of these cells, parafollicularily localized was found in the thyroid gland. An electron dense reaction product was noticed on membraneous structures of C cells of the thyroid.     

Light and immunoelectronmicroscopic study of Hodgkin’s disease: Evidence of immunoglobulin synthesis by tumor cells

The direct immunoperoxidase technique with peroxidase-conjugated F(ab')2 fragments was used at the light and electron microscopic levels to identify intracytoplasmic immunoglobulin (CIg) components in malignant cells of Hodgkin's disease. In each of the 27 cases studied, Hodgkin and Reed-Sternberg cells contained either IgG or IgM, with both light chains often present simultaneously. The number of IgG-positive malignant cells was inversely related to changes in the lymphoid compartment, as defined by the Rye grading system. The evolution from lymphocytic predominance to lymphocytic depletion was paralleled by a decrease of IgM-positive cells and by a substantial increase (to exclusiveness) of IgG-containing cells. These immunoelectronmicroscopic studies disclosed definite morphologic evidence of CIg synthesis by Hodgkin, Reed-Sternberg and lacunar cells. The immunoglobulin components were also synthesized by lymphoid B cells at different levels of modulation. Immunoglobulin synthesis by malignant cells was localized in perinuclear zone, on free cytoplasmic ribosomes and profiles of rough endoplasmic reticulum. The results of this joint light and electron microscopic study support the view that Hodgkin, Reed-Sternberg and lacunar cells belong to the B-cell compartment within Hodgkin's disease.