The histochemical demonstration of guanase: observations in the human central nervous system.

1. A method is described for the histochemical demonstration of the purine catabolizing enzyme guanase, employing glutaraldehyde fixation and Nitro blue tetrazolium (NBT). Parallel biochemical studies confirm that enzyme activity is not significantly inhibited by exposure to glutaraldehyde. 2. By this procedure guanase activity has been visualized in neurons and glial elements of the human central nervous system (CNS). 3. Controls consisted of direct incubation of cryostat sections with a specific inhibitor of guanase (5-amino-4-imidazole carboxamide) and omission successively of the substrate guanine, of xanthine oxidase and of NBT. Enzyme activity was completely inhibited by the above procedures, and by boiling of tissues for 10 min prior to fixation. 4. Levels of enzyme activity in spinal cord and brain were assessed by a subjective scoring method, and showed close comparability with biochemical assay data in brainstem and cerebral hemispheres; whereas a low correlation for enzyme activity was observed in spinal cord and cerebellum. Differences between biochemical and histochemical assessments of CNS guanase activity are discussed.     

Guanosine deaminase in human serum and tissue extracts--a reappraisal of the products

Of the human salvage enzymes that deaminate ribonucleosides, two--cytidine deaminase and adenosine deaminase--have been found particularly useful for diagnostic purposes. In humans, no enzymes are present that can directly deaminate the bases of these ribonucleosides. Indeed, the only enzyme present that can directly deaminate a base is guanine deaminase, and the diagnostic usefulness of this enzyme has been well documented. The aim of this study is to identify the origin of the ammonia formed when human sera and tissue extracts are incubated with buffered guanosine, and to clarify whether the ammonia comes from the deamination of guanosine by guanosine deaminase or is produced as a result of deamination of guanine formed as a breakdown product of guanosine by purine nucleoside phosphorylase (PNP). Apparent deamination of guanosine by guanosine deaminase in human sera and tissue extracts was found to be due to two enzymes acting in tandem when the products of the reaction were examined by HPLC. The ribose was first removed from guanosine by PNP to form guanine, which was then deaminated to xanthine by guanine deaminase.     

Ultrastructural evidence for the synthesis of serum amyloid A protein by murine hepatocytes

After administration of an amyloid-inducing agent to mice, intracytoplasmic localization of serum amyloid A protein in hepatocytes was examined by immunoelectron microscopy. From 6 hours to 14 days after amyloidogenic stimulation, the reaction products were noted mainly on the microvilli and in a few cisternae of Golgi apparatuses. When colchicine was given 3 hours before sacrifice, the reaction products were located in round or oval structures presumed to be the secretory granules derived from the Golgi apparatus and in some autophagosomes. Kupffer cells contained the reaction products during the observation period of 12 hours to 14 days in the phagosomes and on the surface of microvilli or pseudopods but not in the organelles concerned with protein synthesis. These results conclusively support the idea that serum amyloid A protein is synthesized in hepatocytes and that colchicine inhibits the secretion of this protein from hepatocytes into serum.     

Ultrastructure of rat liver cells after exhausting physical training

In rats, running of the maximum intensity caused death of some hepatocytes, an increase in the number of phagosomes in Kuppfer cells, and the emergence of connective tissue fibers in the space of Disse. Ultrastructural investigation of hepatocytes showed delayed release of bile products into bile capillaries, decrease in glycogen content, increase in the number of mitochondria (many of them were divided by the cristae), and irregular distribution of ribosomes in the rough endoplasmic reticulum. Accumulation of erythrocytes in the sinusoids, fragments of dead hepatocytes, Kuppfer cells with numerous phagosomes, and connective tissue fibers in the space of Disse were observed in rat liver after exhausting swimming. Study of hepatocyte ultrastructure revealed intense protein synthesis (as evidenced by increased number of ribosomes and unchanged mitochondria and cisternae of the rough endoplasmic reticulum), separation of cytoplasmic fragments with ribosomes into sinusoids, absence of glycogen, and lipid accumulation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 101–104, July, 1998     

Ultrastructural analysis of hepatitis B surface antigen production in vitro

Recently several continuous cell lines (among these PLC/PRF/5 cells) producing hepatitis B surface antigen (HBsAg) were established from human hepatocellular carcinomas. The cultured cells provide the first opportunity to study HBsAg synthesis and secretion in vitro. HBsAg, but not HBcAg, was localized by the fluorescent antibody technique in the cytoplasm and on the surface of the cultured cells. Under the electron microscope, th PLC/PRF/5 cells displayed morphologic characteristics of both hepatocytes and hepatocellular carcinoma cells. However, 22-nm. spherical or filamentous HBsAg particles were not seen in the cells, although spherical HBsAg particles were observed in the supernatant culture media. Therefore, the indirect immunoperoxidase technique was used to demonstrate HBsAg at the ultrastructural level. Electron-dense reaction product was detected along the nuclear envelope, on rough-surfaced endoplasmic reticulum, and in cisternae of endoplasmic reticulum. These findings suggest that HBsAg is synthesized on rough-surfaced endoplasmic reticulum and transferred into endoplasmic cisternae for processing and secretion. This mode of HBsAg production is identical with that observed in hepatocytes of patients infected with hepatitis B virus. The absence of detectable intracellular HBsAg particles suggests that the cultured cells secrete the particles very rapidly or that they may have a defect in intracisternal packaging of HBsAg into particles.     

Localization of ferritin in human liver diseases studied by immuno-histochemical and immuno-electron microscopic procedures

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.     

LOCALIZATION OF FERRITIN IN HUMAN LIVER DISEASES STUDIED BY IMMUNO-HISTOCHEMICAL AND IMMUNO-ELECTRON MICROSCOPIC PROCEDURES

Localization of ferritin using a pre-embedding diffusion technique and an indirect localization sequence has been made in 34 cases of human liver under normal and several pathological conditions. With light microscopic observation, positive immuno-staining for ferritin was demonstrated as diffuse deposits in the hepatocytes and Kupffer cells. Intensity of the positive immuno-staining for ferritin in these cells appeared to roughly coincide with serum ferritin levels of each patient, but showed no disease specificity, although hepatoma cells contained weak deposits or were negative from immuno-staining for ferritin. With electron microscopic studies, intracellular antigen was well preserved in the hepatocytes and Kupffer cells in most cases with the positive immuno-staining for ferritin being observed in cytosol and a few cisternae of rough endoplasmic reticulum. Content of the positive immuno-staining for ferritin differed considerably from one case to another and one cell to another even in the same case. There was no immuno-staining for ferritin in hemosiderin pigment, lysosome, most of rough endoplasmic reticulum, Golgi complexes, and nucleus in both cells.     

Na-glutamate sensitive neurons in the area postrema of the rat (author''s transl)

A single subcutaneous injection of monosodium-L-glutamate induces severe ultrastructural alterations in certain AChE positive parenchymal cells of the Area postrema of the adult rat. Signs of cellular degeneration include massive intracellular edema, swelling of mitochondria, vacuolization of the cisternae of the rough endoplasmic reticulum and marked alterations in the chromatin pattern of the nucleus. Identification of these cells as neurons is based on the presence of axosomatic synapses.     

Biochemical characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphorybosyltransferase: role of histidine residue in substrate selectivity

The enzyme hypoxanthine-guanine phosphorybosyltransferase (HGPRT) in the malarial parasite Plasmodium falciparum (Pf) is central to the salvage pathway for purine nucleotide biosynthesis and is a potential antimalarial chemotherapeutic target. The pH profile of the enzyme activity using xanthine as a substrate shows the possible involvement of a histidine residue in the activity of the enzyme. Chemical modification studies using diethylpyrocarbonate (DEPC) also corroborate this hypothesis. A comparative sequence alignment of Pf HGPRT with the human, Tricomonus foetus and Toxoplasma gondii HGPRT, coupled with the 3D structural alignment between these enzymes indicated that a histidine residue at position 196 of the Pf HGPRT sequence was located in the close proximity to the active site. Site directed mutagenesis of this histidine residue to lysine (the corresponding residue in the human enzyme) specifically abrogated xanthine and guanine utilization of the enzyme without affecting the conversion of hypoxanthine to its corresponding nucleotide. The mechanism of action for this enzyme was evaluated by steady state kinetics for the substrates xanthine, guanine and PRPP and product inhibition studies. The results indicate the possibility of ping-pong mechanism for the enzyme in contrast to the ternary complex mechanism followed by the human enzyme. These results show that the difference in human and malarial HGPRT can be gainfully exploited to design specific inhibitor for this enzyme.     

Determination of urinary methylated purine pattern by high-performance liquid chromatography

We describe the group selective separation and quantification of unmodified and modified purines in human urine by high-performance reverse phase liquid chromatography. The pattern of oxypurines and methylated purines: hypoxanthine (Hx), xanthine (X), 1-methyl hypoxanthine (1-MHx), 1-methyl guanine (1-MG), 3-methyl guanine (3-MG), 7-methyl guanine (7-MG), 1-methyl xanthine (1-MX), 3-methyl xanthine (3-MX), 7-methyl xanthine (7-MX), 1,7-dimethyl guanine (1,7-dMG), 1,3-dimethyl xanthine (1,3-dMX), 1,7-dimethyl xanthine (3,7-dMX) and 1,3,7-trimethyl xanthine (1,3,7-tMX) were determined in a single run in urine of a healthy subject and a gout patient before and after treatment with allopurinol. This method may be useful to investigate the urinary pattern of methylated bases in diseases involving purine metabolism.     

Alterations in the fine structure of hepatocytes in hyperthyroid rats

Ultrastructural investigation of liver from ten radiothyridectomized adult male albino rats, made hyperthyroid by administration of desiccated thyroid for eight to ten weeks, revealed changes in hepatic organelles, but no differences between centrilobular, midzonal and periportal hepatocytes of a single lobule. The mitochondria were enlarged with an increase in matrix density, but no increase in number of mitochondria or alterations in membranes or criste was observed. The smooth endoplasmic reticulum appeared slightly increased and dilated in treated rats, while stacked cisternae of the rough endoplasmic reticulum were seldom seen. Large vacuoles, which often contained follicular material and frequently opened into the spaces of Disse, were observed at the periphery of hepatocytes. The vacuoles may arise from invaginations of the cell membrane along these spaces to increase the surface area and to act as channels for liver metabolites. Moreover, in hyperthyroid rats hepatic glycogen was uniformly depleted. Whether these changes were a primary effect of thyroid hormone or secondary to metabolic alterations is unclear.     

Ultrastructural hepatic alterations in hamsters and jirds after experimental infection with the liver flukeOpisthorchis viverrini

Changes in the hepatocytes of male hamsters (Mesocricetus auratus) and jirds (Meriones unguiculatus) at 220 days after experimental infection with the liver flukeOpisthorchis viverrini were studied by light and electron microscopy. The hepatocytes of the control group were characterized by an intracellular compartmentation. A globular nucleus was located centrally. The main features of the perinuclear zone were the cisternae of the rough endoplasmic reticulum (RER) and interjacent mitochondria, lysosomes, and peroxisomes. The peripheral cell region was dominated by glycogen fields and scattered lipid droplets, which were surrounded by anastomosing tubules of the smooth endoplasmic reticulum (SER). An immense proliferation of the SER was striking in the hepatocytes of animals infected withO. viverrini. Coincidentally, the intracellular compartmentation disappeared. Glycogen rosettes, RER, lysosomes, and lipid droplets were distributed irregularly all over the cell, the latter being observed more frequently than in control animals. The nuclei showed lobe-like protrusions and were enlarged. The mitochondria were often dumbbell-shaped and showed pathologic degenerations up to lysis. Our results resemble those of numerous investigations concerning hepatocellular alterations caused byN-nitroso compounds. Therefore, these observations suggest a synergistic effect for trematode infection andN-nitroso compounds in the pathogenesis of opisthorchiasis. The cellular alterations observed in the hepatocytes ofOpisthorchis-infected animals together with the accumulation of intermediate filaments seen in the adjacent bile-duct epithelia and in the epithelium of the gallbladder seem to indicate a disturbance of the cell metabolism and might be related to a neoplastic transformation.Abbreviations used in the figures bd bile duct - be bile-duct epithelium - c bile canaliculus - fl fluke - gd Golgi apparatus - gl glycogen - gr granulocyte - H hepatocyte - if intermediate filaments - li lipid droplet - lys lysosome - m mitochondrion - mv microvilli - mvb multivesicular body - n nucleus - nu nucleolus - pc periductual connective tissue - RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum - sg secretory granules - si sinusoid - v vacuole     

Contributions of sodium and chloride to ultrastructural damage after dendrotomy

Summary To determine the contributions of sodium and chloride to ultrastructural changes after mechanical injury, we amputated primary dendrites of cultured mouse spinal neurons in low calcium medium in which sodium chloride had been replaced with either choline chloride or sodium isethionate or sodium propionate. Uninjured cultured neurons were also exposed to the sodium ionophore, monensin. A third set of neurons was injured in medium in which all sodium and calcium chloride had been replaced with sucrose. Neurons injured in low-calcium, low-sodium medium exhibited few ultrastructural changes, except very near the lesion, where there was some dilation of mitochondria and cisternae of the smooth endoplasmic reticulum (SER). Mitochondria in other regions of the neurons developed an electron opaque matrix, and those nearer to the lesion converted to the condensed configuration, characterized by expanded intracristal spaces as well as a dense matrix. If sodium but not chloride was present in the medium, there was some dilation of the Golgi cisternae after injury, as well as some increased electron opacity of the mitochondria. Monensin treated neurons also exhibited dilation of the Golgi cisternae. Neurons injured in sucrose-substituted medium showed none of the changes associated with injury in normal culture medium. These results indicate that sodium influx through the lesion is involved in the dilation of the SER, which is seen even in low-calcium medium, and that a permeant anion, such as chloride, is also involved. This dilation of the SER may result from uptake of calcium released from mitochondria in response to elevated cytosolic sodium. Dilation of the Golgi cisternae appears to be a response only to elevated intracellular sodium. Condensation of the mitochondria after injury is thought to be due to increased demands for ATP synthesis and may involve a futile cycling of calcium across the mitochondrial membrane, involving sodium-mediated calcium release in response to elevated intracellular calcium.     

Fenestrae in the rough endoplasmic reticulum of Xenopus laevis hepatocytes

The rough endoplasmic reticulum (RER) of Xenopus laevis hepatocytes was examined by freeze-fracture and by conventional thin section electron microscopy. Much of the RER was present as stacks of cisternae at the cell periphery but, in addition, large whorls of cisternae were seen in the cytoplasm in most sections. Freeze-fracture replicas revealed fenestrae in both stacked and whorled cisternae, although the fenestrae were more numerous in the whorls. The role of these fenestrae is unknown, but such structures would facilitate access of precursors to the protein synthetic machinery in this highly metabolically active cell type. This would be particularly important in RER whorls, where the innermost cisternae would otherwise be isolated from the rest of the cytoplasm.     

ONO-AE-248诱导的中性粒细胞非凋亡性程序化细胞死亡的形态学变化

目的研究前列腺素E受体亚型3(EP3)激动剂ONO-AE-248诱导的中性粒细胞(PMN)非凋亡性程序化细胞死亡的形态学变化,探讨PMN死亡形式的多样性和复杂性。方法运用透射电镜、荧光显微镜及激光共聚焦扫描显微镜观察PMN在自发性凋亡与ONO-AE-248刺激下亚细胞微结构的变化。结果以5mmol/L的ONO-AE-248刺激PMN12h后,电镜观察绝大多数PMN的多叶核融合为单叶核,核质疏松,核膜分层、起泡甚至破裂,核质溢出,但细胞膜较完整,细胞器无明显改变。以荧光染料DAPI标染活细胞核,ONO-AE-248刺激后,PMN表现为多叶核融合成大而模糊的球形,核染色密度较低;ONO-AE-248刺激的PMN死亡仍有细胞膜磷脂酰丝氨酸(PS)的外翻。ONO-AE-248刺激组与自发性凋亡组PMN在线粒体的形态结构、分布和细胞质内的染色性均有明显差异。结论ONO-AE-248在体外能刺激人PMN发生非凋亡性程序化细胞死亡,这一刺激过程不影响自发性凋亡的PMN细胞膜PS的外翻,并且细胞核和线粒体的改变是其早期最为显著的形态学改变,提示二者可能是ONO-AE-248刺激的靶标和早期的药物反应中心。     

C-reactive protein selectively enhances the intracellular generation of reactive oxygen products by IgG-stimulated monocytes and neutrophils.

The acute phase protein, C-reactive protein (CRP), when heat-aggregated (Agg-CRP), potentiates immunoglobulin G (IgG) Fc receptor-mediated luminol-enhanced chemiluminescence (CL) in human monocytes and neutrophils. Luminol-CL is a sensitive measure of phagocyte respiratory burst activity; however, the nature of oxidative products contributing to the light emission and their site of generation remain incompletely defined. To more precisely describe the oxidative burst of monocytes and neutrophils to Agg-CRP, superoxide anion release was measured by cytochrome c reduction. In addition, the extracellular release of hydrogen peroxide was distinguished from hydrogen peroxide generation using a phenol red oxidation assay. Finally, a flow cytometric determination of dichlorofluorescein (DCFH) oxidation was employed as an index of intracellular peroxide production. Although Agg-CRP alone did not stimulate hydrogen peroxide generation by either monocytes or neutrophils, it significantly enhanced hydrogen peroxide generation in response to heat-aggregated IgG (Agg-IgG). In contrast, Agg-CRP did not enhance the extracellular release of either hydrogen peroxide or superoxide anion from Agg-IgG-stimulated cells. The capacity of Agg-CRP to enhance selectively intracellular oxidative product generation was confirmed when measuring DCFH oxidation in Agg-IgG-stimulated cells. To evaluate whether this selective enhancement of intracellular oxidative events could be attributed, at least in part, to a scavenging effect of Agg-CRP, a cell-free oxygen radical-generating system was employed. Agg-CRP did not significantly diminish the lucigenin-amplified CL response induced by the xanthine/xanthine oxidase reaction. These results indicate that although Agg-CRP enhances the intracellular generation of reactive oxygen intermediates by monocytes and neutrophils, extracellular release of those products is not influenced by cell interaction with Agg-CRP. It is tempting to speculate that CRP can selectively boost the microbicidal activities of monocytes and neutrophils within an inflammatory site by amplifying the intracellular generation of reactive oxygen products without increasing damage to surrounding normal tissues.     

Ultracytochemical charactrization of non-specific acid phosphatase activities in Saccharomyces cerevisiae

Glutaraldehyde prefixation causes a considerable inactivation of the acid phosphatase of yeast protoplasts in dependence on the duration of aldehyde influence. Lead ions necessary for ultracytochemical demonstration effect a still stronger inhibition of enzymatic activity. Prefixation, however, protects the enzyme from further inhibition by lead. At pH 4.4 in intact cells acid phosphatase activities are mainly localized in the periplasmic space and in vesicles fused with the plasma membrane. The cell wall and cytoplasm usually remain free of reaction products. On the cell surface activities are found in form of globular lead deposits. At pH 5.2 and 6.3 the periplasmic activity appears decreased compared to that at lower pH values and the intracellular activity is increased. The plasma membrane of protoplasts is completely free of precipitates. The intracellular activity sites of protoplasts (cisternae of endoplasmic reticulum and/or Golgi-like system, small vesicles, central vacuole, nuclear envelope) are the same as for intact cells. The occurrence of at least two forms of acid phosphatase in S. cerevisiae is deduced.     

IMP dehydrogenase deficiency in Leishmania donovani causes a restrictive growth phenotype in promastigotes but is not essential for infection in mice

Leishmania cannot synthesize purines de novo and therefore must scavenge purines from its host for survival and growth. Biochemical and genomic analyses have indicated that Leishmania species express three potential routes for the synthesis of guanylate nucleotides: (1) a two-step pathway that converts IMP to GMP; (2) a three-step pathway that starts with the deamination of guanine to xanthine, followed by phosphoribosylation to XMP and then conversion to GMP; or (3) direct guanine phosphoribosylation by HGPRT. To determine the role of the first of these pathways to guanylate nucleotide synthesis, an L. donovani line deficient in IMP dehydrogenase (IMPDH), the first step in the IMP to GMP pathway, was constructed by targeted gene replacement. The Δimpdh lesion triggered a highly restrictive growth phenotype in promastigotes in culture but did not impact parasitemias in mice. The dispensability of IMPDH in vivo is the first definitive demonstration that intracellular L. donovani amastigotes have access to a sufficient pool of guanine, xanthine, or guanylate precursors from the host.     

Chemical composition of some components of the arrestment pheromone of the black-legged tick, Ixodes scapularis (Acari: Ixodidae) and their use in tick control

Chemical analysis (high-performance liquid chromatography) and bioassay demonstrated the presence of compounds that seem to be components of the Ixodes scapularis arrestment pheromone. Only two purines, guanine and xanthine, were found in acidified saline extracts made from cast skins after molting of fed nymphs, fed larvae, and fecal/excretory exudates deposited by unfed adults on substrates in their environment. The ratio of guanine to xanthine was 10.6:1 in an extract from the nymphal skins versus 0.95:1 in an extract from the larval skins. Guanine, xanthine, and traces of a third purine, tentatively identified as 8-azaguanine, were found in extracts made from filter paper strips or washings from glass vials contaminated with tick feces and excreta left by unfed adults. 8-azaguanine may be a product of microbial degradation of the other purines rather than a natural product from the ticks. Low concentrations of ammonia also were detected in saline extracts of excreta from feeding ticks. Hematin also was found in NH4OH extracts of the black fecal/excretory exudates deposited by the unfed ticks. Hematin was tentatively identified by comparison of spectra with that of the authentic standard. Bioassays demonstrated a strong positive arrestment response to cast skins found to contain a mixture of guanine and xanthine and to black fecal/excretory exudates containing guanine, xanthine, the putative 8-azaguanine, and hematin. A Noldus video tracking system using a CCD video camera and Ethovision Pro tracking software showed statistically significant increases in the frequency of visits to the treated zone versus the control. Ticks were significantly more likely to assemble in response to the tick exudates within as little as 3 h compared with the controls. Previous bioassay studies also showed strong positive responses to guanine, xanthine, other purines, and hematin. Comparisons with the arrestment pheromones of other tick species are described. The inclusion of the pheromone components in a permethrin-impregnated oily matrix, Last Call, increased the lethal activity of the product to 95% compared with only 65% in the formulation with permethrin alone. More detailed knowledge of I. scapularis arrestment pheromone may be useful for improving the efficacy of this tick-killing technology even further.     

Synaptic and Golgi membrane recycling in cochlear hair cells

Summary Membrane recycling in the mechanoreceptive sensory cells of the mammalian cochlea was studied by observing membrane-bound horseradish peroxidase (HRP) reaction product following briefin vivo exposure to the enzyme. In the inner hair cell (IHC), peroxidase was taken up into coated vesicles and became incorporated into synaptic vesicles surrounding presynaptic bodies, but much HRP was also transported to the apical zone where reaction product appeared in all components of the Golgi complex. Neither the subsurface cisternae nor a tubular network associated with clusters of mitochondria were labelled. Outer hair cells (OHCs) showed considerably less membrane-bound reaction product than IHCs, indicating less rapid plasmalemmal recycling. Most membrane-bound reaction product was contained in coated vesicles and small vacuoles in the synaptic zone, but was occasionally seen in multivesicular bodies in the most apical zone. No labelled organelles were detected in the large central region of the OHC. A diffuse staining of the cytoplasm, particularly pronounced in OHCs, often interfered with the evaluation of membrane-bound reaction product in OHCs. This staining pattern could be qualitatively reproduced in both IHCs and OHCs by incubating fixed segments of the organ of Corti in oxidized diaminobenzidine. The presence of labelled synaptic vesicles associated with presynaptic bodies of IHCs and OHCs suggests that they are formed from membrane retrieved from the plasmalemma. We found no evidence that the subsurface cisternae of IHCs or the laminated cisternae of OHCs are derived from the cell surface as they never contained reaction product.