Mucosubstances of rabbit granulocytes studied by means of electron-microscopic radioautography and X-ray microanalysis.

Rabbit bone marrow cells have been studied by means of light- and electron-microscopic radioautography and X-ray microanalysis. Carrier free sulfuric acid was used as the radioactive precursor in this experiment. Immature granulocytes showed more active incorporation than mature ones. Silver grains were observed in the Golgi apparatus and granules in three kinds of granulocytes. Electron-microscopically, immature granules showed the incorporation of inorganic sulfate, while mature ones did not. Sulfur was detected in all kinds of granules of the three granulocyte types by X-ray microanalysis. It is concluded that incorporated inorganic sulfur may be utilized for the synthesis of acid glycosaminoglycans and the sulfur detected by X-ray microanalysis may be that contained in the acid glycosaminoglycan. The sulfur detected in the specific granules of the heterophil probably derives from proteins or polypeptides incorporating sulfur containing amino acids.     

Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.     

Selective digestion of mouse chromosomes with restriction endonucleases. II. X-ray microanalysis of HaeIII-treated chromosomes

We used X-ray microanalysis to study the changes induced in mouse metaphase chromosomes as a result of digestion with the restriction endonuclease HaeIII. The phosphorus X-ray signal was used as a marker for DNA and the sulfur signal for protein. Calcium, iron, copper, and zinc were also detected. HaeIII induced a loss of phosphorus from both the centromeres and chromosome arms, but the losses in the arms were much greater. These changes were accompanied by an increase in the electron density of the centromeres and a reduction in that of the arms. No reduction in the sulfur signal in either arms or centromeres occurred as a result of HaeIII digestion. Except for calcium, which showed only a moderate reduction, the inorganic ions exhibited very large losses as a result of HaeIII digestion. The differentiation of chromosome arms and centromeres as a result of HaeIII digestion is therefore not simply due to differential loss of DNA but also involves structural reorganization of the chromatin, as shown by electron microscopy. This reorganization does not involve loss of proteins but may be correlated with changes in the amounts of inorganic ions known to be involved in chromatin condensation.     

Electron microscopy of human melanosomes in unstained,fresh air-dried hair bulbs and their examination by electron probe microanalysis

Summary Electron microscopy of unstained, fresh air-dried spreads of plucked human hair bulbs revealed relatively well preserved melanocytes and dense storage granules of melanin. Energy dispersive X-ray microanalysis of melanosomes and premelanosomes from black facial hair bulbs disclosed high peaks for potassium and sulfur, intermediate peaks for magnesium and calcium, and low but distinct ones for phosphorus and chlorine. No peaks for magnesium were found in the central portion of mature melanosomes, but at their edges and in premelanosomes conspicuous peaks for this element were obtained.Supported by a grant from the Ministry of Education of JapanThe author thanks Dr. H. Fujita for his advice and Messrs K. Yonehara, S. Okamura, T. Fukuoka, and T. Asai, Naka Works, Hitachi Ltd., Katsuta, Ibaraki, for use of their X-ray microanalyzer and for their assistance in electron probe analysis     

Energy dispersive X-ray microanalysis of neurosecretory granules of mouse pituitary on fresh air-dried tissue spreads

Electron microscopy of fresh air dried spreads of unstained posterior lobe tissue from mouse pituitary disclosed neurosecretory granules. Each granule showed a seemingly homogeneous dense core surrounded by a halo and a bounding membrane. The area between granules in the cytoplasm was relatively well preserved. The energy dispersive X-ray microanalysis revealed peaks for sulfur, chlorine and potassium in two granules. The third granule displayed peaks for phosphorus and chlorine. These elements probably contribute to the high electron density of the granules. There was no peak for calcium, in contrast to the dense bodies of human blood platelets.     

Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte.     

A comparative study of primary and secondary granules in monocytopoiesis and myelopoiesis of mouse bone marrow

Summary The differentiation and maturation of monocytes and neutrophil granulocytes were studied in bone marrow of normal mice by electron microscopy and cytochemical assessment of peroxidatic activity. The granule populations of the mature cells of bone marrow were identified and investigated to obtain a basis for the analysis of the earlier stages of maturation. Mature monocytes and neutrophils showed primary and secondary granules, and mature neutrophils had more of both kinds. The size, shape, and number of primary granules proved to offer the most reliable criteria for distinguishing promonocytes and promyelocytes. The primary granules of monocytes were smaller than those of mature neutrophils and were either spherical (smallest diameter 50–200 nm) or elongate (100×400 nm). Both granules had a homogeneous matrix. The granules of the granulocytes were either spherical (smallest diameter 200–300 nm) or elongate (150–200×300–500 nm), and some of them had a crystalline inclusion.     

Intranuclear silicon detection in a subcutaneous connective tissue cell by energy-dispersive x-ray miscroanalysis using fresh air-dried spread.

Silicon was detected by energy-dispersive x-ray microanalysis in the nucleus of a subcutaneous connective tissue cell of mice fed normally. To eliminate contamination, pieces of connective tissue were spread on copper grids and examined without any treatment by an energy-dispersive spectrometer with a scanning transmission apparatus attached to an electron microscope. Scanning transmission electron microscopy of the spread has demonstrated a well-preserved ultrastructure. Fibrous structures, nuclei and nucleoli of cells and mitochondrial granules were recognized. Electron probe analysis showed peaks for silicon at three spots on the nucleus of a cell in addition to those for phosphorus, sulfur, chlorine, potassium and calcium, whereas no peak of silicon could be detected at the spots on nuclei of other cells, mitochondrial granules and electron-lucent area on the same grid as the above. Silicon appears to play a significant role in the nucleus. Applicability of the technique to know the distribution of easily contaminating elements and diffusible substances is shown.     

The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions.     

Immunohistochemical characterization of purple acid phosphatase-containing leucocytes in the human placenta

Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.     

Characteristics of the granules formed in nucleated erythrocytes as affected by cardiotrast

Numerous granules are formed in frog erythrocytes under the influence of cardiotrast (C) (diethanolamine-3,5-diiodo-4-pyridone-1-acetic acid). However, as revealed by cytospectrophotometric investigation and X-ray microanalysis, no C was accumulated in these granules. It is known that C can dissociate into diethanolamine and 3,5-diiodo-4-pyridone-1-acetic acid. It was assumed that under the influence of C granule formation may occur at the expense of an uncharged diethanolamine form penetrating into lysosome-like structures to be accumulated by protonation process. Diethanolamine was found to provoke granule formation in frog erythrocytes. It is impossible to reveal substance in granules because it is colourless and has no ultraviolet absorbtion band. Under the influence of some inhibitors of energy metabolism on granule formation and the granules formed, their inhibitory effect is exerted on the process of granule formation. In granules isolated by differential centrifugation activity of some marker lysosomal enzymes was found which enabled us to attribute these granules to lysosome-like structures.     

The composition of pyrophosphate heavy metal detoxification granules in barnacles

A major detoxification mechanism involving the binding of excess metals to inorganic, insoluble metalliferous granules occurs in many invertebrates. X-ray microanalysis of granules extracted from the barnacles Elminius modestus Darwin and Semibalanus balanoides (L.) (Crustacea: Cirripedia) indicated that one granule type was predominant. Examples of this granule type usually contained Fe and P, or Zn and S, or a combination of all four elements as major constituents. The predominant form of P in the granules was pyrophosphate, P₂O⁴⁻₇, with oprthophosphate and other polyphosphates present in lower amounts. The S was present as organic S. Biochemical analysis showed high levels of Ca, Fe, Mg, K and Zn in the granules with Cu and Ag present in smaller quantities. Considerable variation was shown between granules.     

Immunogold localisation of ubiquitin-protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils.

Ubiquitin-protein conjugates are found in the primary (azurophilic) lysosome-related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin-protein conjugates in lysosome-related membrane-bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin-protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.     

Specific granules of human eosinophils have lysosomal characteristics: presence of lysosome-associated membrane proteins and acidification upon cellular activation

Eosinophils possess characteristic specific granules. Their content may be important during host defense but it can also cause damage after release at sites of inflammation. We investigated possible lysosomal characteristics of these granules. Lysosome-associated membrane protein (LAMP)-1 and 2, were detected by Western blot, subcellular fractionation, and immunoelectron microscopy (IEM) and were localized to the membrane of specific granules and in vesicles of the cytoplasm, separate from secretory vesicles. No binding of mannose 6-phosphate receptor to proteins of specific granules could be detected, indicating that they are dephosphorylated and mature. Cellular activation by interleukin-5 caused acidification of specific granules, as detected by pH-dependent probes. The acidification was inhibited by concanamycin A (inhibitor of vacuolar H(+)-ATPase). Activation of eosinophils by serum-treated zymosan (STZ) caused degranulation into STZ-containing phagosomes and incorporation of LAMPs to their membranes. In conclusion, specific granules of eosinophils can be regarded as specialized primary lysosomes, a feature that may be important for their function and integrity.     

Differentiation of eosinophilic granulocytes of carp (Cyprinus carpio L.).

Electron-microscopic studies were conducted to observe ultrastructural changes during differentiation of eosinophilic granulocytes in carp (Cyprinus carpio L.). Differentiation at the myelocyte stage was found to relate to specific granules made of dense and light fields. By maturation they assume a mosaic-like texture and in each granule of mature granulocytes, a light, central "internum" and a peripheral dense wrapper can be distinguished. The activity peroxidase and acid phosphatase is located in the internum and of peroxidase in the wrapper of the granules.     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Identification of Normal and Leukemic Granulocytic Cells with Merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytos and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in crythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single a p t stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.     

Method for the Prfservation of Polystyrene Latex Beads in Tissue Sections

The degree of infiltration of epoxy resin into pituitary secretory granules was evaluated using X-ray microanalysis of the concentrations of chlorine in the epoxy resins. The effectiveness of infiltration was tested after three different tissue preparation techniques: cryofixation + freeze-drying (CF-FD), glutaraldehyde fixation (GF) + chemical dehydration, and no fixation— no dehydration. Signs of marked incomplete infiltration were found in embedded unfixed tissue while the other two techniques showed 80% infiltration. Uneven penetration was seen after CF-FD and GF. The plastic surface demonstrated a mountain-like appearance over the secretory granules after immunocytochemistry of the glutaraldehyde fixed tissue, whereas the CF-FD tissue showed a less furrowed surface. This probably is due to contact with water, which swells those parts of the granules that are unprotected by the plastic embedding medium. Our findings may explain why it is possible to perform immunocytochemistry on Epon embedded tissue.     

X-ray micronalysis of copper and sulphur-containing granules in the fat body cells of homopteran insects

Regions of the fat body of larvae of Chaetophyes compacta and Pectinariophyes sp. (Machaerotidae, Homoptera) which are closely associated with mycetomes have been analysed by electron probe X-ray microanalysis. It is shown that cells in these regions contain electron probe X-ray microanalysis. It is shown that cells in these regions contain electron dense granules which are rich in copper and sulphur. These two elements occur in the atomic ratio of 3:2 respectively. It is conjectured that copper may be bound to a sulphur containing metallothionein and that the granules represent either the end products of copper detoxification or serve as copper stores for synthesis of enzymes and macromolecules by the mycetomal symbionts.