The human chromophobe cell renal carcinoma: its probable relation to intercalated cells of the collecting duct

In the present study we have examined ten cases of the chromophobe type renal cell carcinoma. This type of tumor is distinguished from the other carcinomas of the kidney with light cytoplasm (formerly called "hypernephroid") by (a) a positive Hale's iron colloid stain of the cytoplasm, (b) the occurrence of numerous invaginated vesicles within the cytoplasm that resemble the invaginated vesicles of intercalated cells of the collecting duct system, and (c) a positive immunoreaction of both the plasma membrane and the cytoplasm with antibodies to the epithelial membrane antigen (EMA) and carbonic anhydrase C (CAC), respectively. Unlike oncocytomas, which also express CAC and EMA, the chromophobe renal cell carcinoma does not express the erythrocyte anion exchanger band 3. These findings strongly indicate that chromophobe renal cell carcinomas as well as oncocytomas of the kidney are histogenetically related to the two populations of intercalated cells of the collecting duct system. Thus, both tumors represent examples of renal tumors which disprove the broadly accepted hypothesis that all epithelial tumors of the kidney are histogenetically related to the proximal tubule.     

Thin section and freeze fracture studies of the hypophyseal proximal pars distalis in a teleost (Rhamdia hilarii Val.) during different stages of the reproductive cycle

Summary Chromophilic cells in the proximal pars distalis of the adenohypophysis of Rhamdia hilarii were studied in thin section and freeze fracture preparations. The gonadotropic cells (GTH-cells) exhibit a diversity of form, the frequency of which can be related to stages (maturation, mature and spent) in the sexual cycle. GTH-cells showing a cytoplasm filled with electron dense polymorphic secretory granules and small rough endoplasmic reticulum (RER) vesicles, have been termed non-vacuolated. During the mature gonadal stage, such cells become increasingly vacuolated. The small RER vesicles become dilated and/or fuse, forming a single enormous cisternum (4–11 m diameter), the contents of which show direct contact with the inner nuclear membrane. These morphological aspects support the idea that Rhamdia hilarii possesses only one GTH-cell type. Evidence from freeze fracture replicas suggests that membrane-associated events precursory to exocytosis take place in regions where the cell and secretory granule membranes are in close apposition. Thin section analysis of secretory granule formation revealed their derivation from the dilated extremities of the inner Golgi saccule which appears to resemble the rigid lamella described in other cells. After detachment of the inner saccule, the immature secretory granules appear to enlarge by microvesicular transport. Freeze fracture and ultrastructural data on the morphology of the cells that presumably synthetise growth hormone are also presented.This work was aided by the Fundação de Amparo à Pesquisa do Estado de São Paulo (75/1282)     

Origin of the membrane for the acrosomal process: is actin complexed with membrane precursors?

The formation of the acrosomal process requires an increase of 40-150% in the plasma membrane limiting the cell body. In freeze fracture replicas and thin sections this new membrane behaves as a typical unit membrane. Since in sperm there are no vesicles or surface projections which could account for the increase in surface, we suggest that the new membrane is generated from precursors stored with the actin in the periacrosomal region. Consistent with this suggestion is the observation that compounds such as didansyl cysteine and N phenyl-1-napthylamine which fluoresce when they enter a hydrophobic environment stain the periacrosomal material.     

Is lipid translocation involved during endo- and exocytosis?

Stimulation of the aminophospholipid translocase, responsible for the transport of phosphatidylserine and phosphatidylethanolamine from the outer to the inner leaflet of the plasma membrane, provokes endocytic-like vesicles in erythrocytes and stimulates endocytosis in K562 cells. In this article arguments are given which support the idea that the active transport of lipids could be the driving force involved in membrane folding during the early step of endocytosis. The model is sustained by experiments on shape changes of pure lipid vesicles triggered by a change in the proportion of inner and outer lipids. It is shown that the formation of microvesicles with a diameter of 100-200 nm caused by the translocation of plasma membrane lipids implies a surface tension in the whole membrane. It is likely that cytoskeleton proteins and inner organelles prevent a real cell from undergoing overall shape changes of the type seen with giant unilamellar vesicles. Another hypothesis put forward in this article is the possible implication of the phospholipid 'scramblase' during exocytosis which could favor the unfolding of microvesicles.     

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.     

Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogense, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

William Trager: An Appreciation

SYNOPSIS. Additional information on host interactions with trypanosomatid membranes was obtained from studies of a monomorphic strain of Trypanosoma brucei harvested at peak parasitemia from intact and lethally irradiated rats. Pellets of trypanosomes were fixed briefly in glutaraldehyde and processed for thin section electron microscopy or freeze-cleave replicas. Observations of sectioned material facilitated orientation and comparison of details seen in replicas. Fracture faces of cell body and flagellar membranes as well as 3-dimensional views of the nuclear membrane were studied. Cell body membranes of 80% of the organisms from intact rats contained random arrays of intramembranous particles (IMP). Aggregated clusters of particles appeared on the fracture faces of 20% of the trypanosomes. Some of these membranes had nonrandomly distributed particles aligned in distinct rows on the outer fracture face of both cell body and flagellum. Many inner face fractures of the cell body membranes had a particle arrangement similar to the longitudinal alignment of cytoskeletal microtubules. No aggregated particle distribution was seen in membranes of trypanosomes harvested from lethally irradiated rats. Replicas of trypanosome pellets also had plasmanemes as a series of attached, empty, coated membrane vesicles. These structures were found in close association with, as well as widely separated from the parasites. The shedding of these vesicles and the variation of particles in cell body membranes are discussed in light of antibody-induced architectural and antigenic changes in surface properties of trypanosomatids. The convex face of the inner membrane of the nucleus also is covered with randomly arrayed particles. More IMP were observed on the inner than on the outer nuclear membranes. Images of nuclear pores were also seen. The importance of these structures in drug and developmental studies of trypanosomes is discussed. On fracture faces of the flagellar membrane there were miniature maculae adherentes, unique to the inner fracture face and occurring only at regions of membrane apposition between cell body and flagellum. Each cluster of particles exposed by the freeze-cleave method corresponds to an electron-dense plaque seen in thin section images. However, because of a unique fracture pattern, these plaques were not revealed on the apposing body membranes, as illustrated in thin sectioned organisms.     

Membrane associations between subsurface cisternae of endoplasmic reticulum and the plasma membrane of rat Sertoli cells

Close membrane associations between the endoplasmic reticulum and the plasma membrane (ER-PM) occur in specialized regions of the rat Sertoli cell cytoplasm. They are characterized, in freeze fracture replicas, as mesa-like modifications of E membrane fracture faces or as corresponding discoid depressions on P membrane fracture faces. When these structures lie along transitional regions in the membrane fracture plane, they are seen to be complementary, and the space between them to be greatly reduced. These specialized close membrane associations may represent adhesive sites between the endoplasmic reticulum and the plasma membrane. However, their resemblance to vascular endothelial fenestrae which are known to be sites of increased membrane permeability may suggest other functional roles.     

Review of chromophobe renal cell carcinoma with focus on clinical and pathobiological aspects

In recent years, the concept of chromophobe renal cell carcinoma (RCC) has been established. Chromophobe RCCs account for about 4-6% of all renal tumors. Macroscopically, the cut surface of the tumor is generally grey-beige in color. Histologically, there are two variants (typical and eosinophilic). In the typical variant, large tumor cells with architecture of a compact tubulo-cystic pattern proliferate. The cytoplasm is abundant and shows a fine reticular translucent pattern. The cell border is thick, prominent and eosinophilic. In the eosinophilic variant, tumor cells are smaller and markedly eosinophilic, and a perinuclear halo is often seen. Histochemically, the tumor cells generally show a diffuse and strong reaction for Hale's colloidal iron staining. Ultrastructurally, tumor cells contain many cytoplasmic microvesicles (150-300 nm). In chromosomal analysis, a low chromosome number is characteristic of chromophobe RCCs, due to the frequent occurrence of a combined loss of chromosomes 1, 2, 6, 10, 13, 17, and 21. In differential diagnosis, histological distinction from oncocytomas, which share a common phenotype (intercalated cells of the collecting duct system), is most important. In this diagnostic setting, recent studies have given rise to several problems. Firstly, some cases of coexistent chromophobe RCC and oncocytoma (so-called renal oncocytosis) or cases of oncocytoma with metastasis have recently been reported. Secondly, the existence of chromophobe adenoma, which is the benign counterpart of chromophobe RCC, and an oncocytic variant of chromophobe RCC has recently been suggested. Therefore, further studies are needed to elucidate the relationship between chromophobe RCCs and oncocytomas, to confirm whether chromophobe adenoma actually exists or not, and to identify the key gene that causes chromophobe RCCs.     

Leishmania mexicana: distribution of intramembranous particles and filipin sterol complexes in amastigotes and promastigotes

The density and distribution of intramembranous particles was analyzed in freeze fracture replicas of the plasma membrane of amastigotes, and infective as well as noninfective promastigotes of Leishmania mexicana amazonensis. The density of intramembranous particles on both protoplasmic and extracellular faces was higher in infective than in noninfective promastigotes and it was lower in amastigotes than in promastigotes. Amastigotes purified immediately after tissue homogenization were surrounded by a membrane which corresponded to the membrane which lined the endocytic vacuoles where the parasites were located within the tissue macrophages. Aggregation of the particles was seen in the flagellar membrane at the point of emergence of the flagellum from the flagellar pocket. Differences in the organization of the particles were seen in the membrane which lined the flagellar pocket of amastigotes and promastigotes. The polyene antibiotic, filipin, was used as a probe for the detection of sterols in the plasma membrane of L. m. amazonensis. The effect of filipin in the parasite's structure was analyzed by scanning electron microscopy and by transmission electron microscopy of thin sections and freeze fracture replicas. Filipin sterol complexes were distributed throughout the membrane which lined the cell body, the flagellar pocket, and the flagellum. No filipin sterol complexes were seen in the cell body-flagellar adhesion zone. The density of filipin sterol complexes was lower in the membrane lining the flagellum than in that lining the cell body of promastigotes.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.     

Fine Structure of Botrytis fabae Sardina Conidia

The fine structure of Botrytis fabae conidia was studied usinga variety of electron-microscope techniques. The spore walllacks conspicuous ornamentation and consists of microfibrilsembedded in a granular matrix. The two distinct wall layersseen in chemically fixed sections cannot be detected in cross-fracturedreplicas; the two layers are probably structurally similar.The outer surface of the plasmalemma is covered with branchedinvaginations and two kinds of particles. Three distinct typesof particles are present on the inner surface of the plasmalemma.In freeze-etched replicas nuclei, vacuoles, and other organellesalways appear smoothly rounded. Small vesicles pass throughthe plasmalemma into the cell wall. Particles approximately10 nm in diameter occur in compact rows on the cristae of cross-fracturedmitochondria: dense spherical particles, probably of calciumphosphate, are present in chemically fixed mitochondria. Prevacuolesand vesicles with membranous inclusions can be seen in bothcross-fractured replicas and chemically fixed sections. In cross-fracturedreplicas vacuoles and lipid bodies are frequently joined bystrands of endoplasmic reticulum.     

Retrieval of lysosomal membrane and acid phosphatase from phagolysosomes of Paramecium caudatum

Little is known about the fate of lysosomal membrane in phagocytic cells. Because the age of the digestive vacuoles in Paramecium caudatum can be easily determined, we have been able to study the dynamic membrane events in the older vacuoles. Late in the phagolysosomal stage (DV-III) the vacuole membrane undergoes a burst of tubule formation. The tubules expand into vesicles which have characteristics resembling lysosomes in both thin sections and freeze-fracture replicas. The tubules also contain acid phosphatase activity when they arise from acid phosphatase-reactive vacuoles. We conclude that after active digestion lysosomal membrane is retrieved in whole or in part along with some membrane-associated hydrolases. A logical extension of these results is that the lysosome-like vesicles, after being recharged with hydrolases by fusing with primary lysosomes, are recycled back to DV-II for reuse.     

Homofusion of Golgi secretory vesicles in flax phloem fibers during formation of the gelatinous secondary cell wall

The gelatinous type of secondary cell wall is present in tension wood and in phloem fibers of many plants. It is characterized by the absence of xylan and lignin, a high cellulose content and axially orientated microfibrils in the huge S2 layer. In flax phloem fiber, the major non-cellulosic component of such cell walls is tissue-specific galactan, which is tightly bound to cellulose. Ultrastructural analysis of flax fiber revealed that initiation of gelatinous secondary cell wall formation was accompanied by the accumulation of specific Golgi vesicles, which had a characteristic bicolor (dark-light) appearance and were easily distinguishable from vesicles made in different tissues and during the other stages of fiber development. Many of the bicolor vesicles appeared to fuse with each other, forming large vacuoles. The largest observed was 4 mum in diameter. Bicolor vesicles and vacuoles fused with the plasma membrane and spread their content in a characteristic "syringe-like" manner, covering a significant area of periplasm and forming "dark" stripes on the inner wall surface. Both Golgi derivatives and cell wall layers were labeled by LM5 antibody, indicating the presence of tissue- and stage-specific (1-->4)-beta-galactan. We suggest that this specific type of galactan secretion, which allows coverage of a large area of periplasm, is designed to increase the chance of the galactan meeting the cellulose microfibrils while they are still in the process of construction. The membrane fusion machinery of flax fiber must possess special components, which may be crucial for the formation of the gelatinous type cell wall.     

Light and electron microscopic immunocytochemical localization of clathrin in rat cerebellum and kidney

The precise cellular and subcellular locations of coated vesicle protein, clathrin, in rat kidney and cerebellum have been visualized by immunocytochemical techniques. In the renal tubular epithelia, clathrin-positive products were found on both free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No clathrin was observed in the cisternae of RER or the Golgi apparatus. Clathrin-positive reaction products could also be seen on coated pits, coated vesicles, Golgi-associated vesicles, basolateral cell membrane, the ground substance, and in the autophagic vacuoles. In cerebellar Purkinje and granule cell bodies, reaction products were seen localized on coated vesicles, on the budding areas from the Golgi-associated membrane and Golgi-associated vesicles. Furthermore, the membrane of the multivesicular body, the bound-ribosomes, and the ground substance were also stained. In the myelinated axon, the clathrin appeared to be concentrated on certain segments and seemed to fill in the space between neurotubules and some vesicles. In certain synaptic terminals clathrin was often seen attached to presynaptic vesicles, presynaptic membrane, and post-synaptic membrane. However, in most mossy fibers, some synaptic vesicles were not stained. These observations suggest that clathrin is synthesized on bound and free ribosomes and discharged into the cytosol where it becomes associated with a variety of ground substances and assembles on coated pits, coated vesicles, Golgi-associated vesicles, presynaptic vesicles, and pre- and postsynaptic membranes. Clathrin may be finally degraded in autophagic vacuoles.     

Fine needle aspiration of chromophobe renal cell carcinoma

OBJECTIVE: To describe the cytomorphologic findings of chromophobe renal cell carcinoma (CRCC) in order to preoperatively distinguish this rare neoplasm from other primary or secondary tumors arising from the kidney or presenting as retroperitoneal masses. STUDY DESIGN: Clinical data, fine needle aspiration (FNA) and follow-up surgical specimens from 4 patients with CRCC (3 primaries and 1 metastatic to the liver) were reviewed. Electron microscopy was available for 2 histologic specimens. RESULTS: Two tumors (1 primary and 1 metastatic case) were readily identified as CRCC on FNA. The 2 remaining cases were diagnosed as renal cell carcinoma (RCC) consistent with CRCC. All tumors showed aspirates with moderate to high cellularity, with the cells arranged in small clusters and single cells. Neoplastic cells had abundant heterogeneous cytoplasm, a thickened cell membrane, nuclear hyperchromasia, nuclear outline irregularity, significant nuclear size variation, intranuclear inclusions and frequent binucleation. Histology of the 4 renal tumors was characteristic of CRCC, with positivity for Hale's colloidal iron in all cases. Ultrastructurally, characteristic cytoplasmic microvesicles were observed in the 2 cases that we studied. CONCLUSION: In the adequate clinicoradiologic setting, CRCC has distinctive cytologic features that may allow an accurate preoperative FNA diagnosis.     

Cell organization and ultrastructure of a magnetotactic multicellular organism

Magnetotactic multicellular aggregates and many-celled magnetotactic prokaryotes have been described as spherical organisms composed of several Gram-negative bacteria capable to align themselves along magnetic fields and swim as a unit. Here we describe a similar organism collected in a large hypersaline lagoon in Brazil. Ultrathin sections and freeze fracture replicas showed that the cells are arranged side by side and face both the external environment and an internal acellular compartment in the center of the organism. This compartment contains a belt of filaments linking the cells, and numerous membrane vesicles. The shape of the cells approaches a pyramid, with the apex pointing to the internal compartment, and the basis facing the external environment. The contact region of two cells is flat and represents the pyramid faces, while the contacts of three or more cells contain cell projections and represent the edges. Freeze-fracture replicas showed a high concentration of intramembrane particles on the edges and also in the region of the outer membrane that faces the external environment. Dark field optical microscopy showed that the whole organism performs a coordinated movement with either straight or helicoidal trajectories. We conclude that the organisms described in this work are, in fact, highly organized prokaryotic multicellular organisms.     

Connections between the cristae and the surface of rat heart muscle mitochondria

Contrary to the generally accepted rule that there are only two fracture faces associated with a membrane, the analysis of double replicas at rat heart muscle mitochondria revealed three pairs of complementary replicas with one face in each pair exposing the outer surface membrane. The replicas must then expose the surfaces of the outer surface membrane and in two of the pairs the fracture had passed between the two surface membranes in two alternative ways, either clearly between the two membranes or the fracture deviated into and through the inner surface membrane at regularly spaced intervals. This deviation reveals that at these sites the connection between the two surface membranes is particularly firm. The analysis led to the conclusion that these sites correspond to those where the stalk-like connections extending from the cristae are connected to the inner surface membrane. This way proteinaceous pathways connect the cristae to the surface of the mitochondria.     

Isolation and electrical characterization of tonoplast vesicles from the Kiwi fruit (Actinidia chinensis)

Nomarski interference microscopy technique showed that the cell juice of the Kiwi fruit ( Actinidia chinensis Planch.) is rich in membrane vesicles that resemble protoplasts and free vacuoles. These vesicles are obtained without enzyme or chemical treatment and probably arise from the rupture and revesiculation of the tonoplasts that limit the cytoplasmic strands of the cells. Vacuole fragmentation in situ probably causes the tonoplast to recombine around the vacuolar sap as well as around the cytoplasmic strands, which implies either original or inverse orientation of the inner face. Electrophysiological measurements in vesicles judged to have the original membrane orientation showed that their polarization was inside positive, the same as central vacuoles of protoplasts and isolated vacuoles.