A method to preserve extracellular surfactant-like phospholipids on the luminal surface of rodent gastric mucosa.

Previous results from our laboratory employing the phospholipid-selective cytochemical stain iodoplatinate (IP) suggest that surfactant-like phospholipids (SLPL) are intracellularly contained within rodent gastric mucous cells and are occasionally seen extracellularly within the mucous gel layer. This hydrophobic lipid coating may provide the stomach with a protective water-repellent lining against the corrosive gastric juice in the lumen. Extracellular SLPL are frequently removed during tissue processing for electron microscopy. In this study, we developed a simple method employing an agarose-embedding technique to retain these extracellular SLPL in gastric mucosa excised from rats pre-treated with prostaglandin (to stimulate gastric surfactant/mucus secretion). With the help of polypropylene supporting screens and cassette carriers, thin slices of agarose-embedded gastric mucosa were well preserved and uniformly stained with IP. Extracellular myelin figures were well retained over the interfoveolar surface as well as in the pit region. The IP-reactive substances were seen within or coating the surface of the mucous gel. Our results also indicate that agarose is useful not only for supporting soft tissue while preparing sections with a microslicer but also for preservation of extracellular lipoidal material for electron microscopic observation.     

Immunoreactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcers

Summary The present study was aimed at assessing whether epidermal growth factor (EGF) and its receptors are present in the gastric mucosa during the healing of gastric ulcers. Immunohistochemical, immunochemical and functional studies were performed in rats after induction of ulcers in the oxyntic mucosa. Controls, which included non-operated and sham-operated animals, displayed only rare cells in the bottom of the oxyntic glands showing EGF-like immunoreactivity. Within one day after ulcer induction, a markedly increased number of chief cells in undamaged mucosa showed intense staining. Concomitantly, there was an increased immunoreactivity for EGF receptors in the mucous neck cells. Maximal immunostaining for both compounds was observed at 3 days after ulcer induction; augmented staining was still demonstrable after 3 weeks. RIA revealed significantly increased EGF concentration in the oxyntic mucosa three days after ulcer induction, and at this stage stimulated gastric acid secretion, measured in a parallel group of chronic fistula rats, indicated significant inhibition. The transient increases in EGF-like and EGF receptor immunoreactivities may stimulate gland cell proliferation. The local release of EGF-like substances may also serve to reduce gastric acidity and thereby promote ulcer healing.     

Stimulant effect of nitric oxide generator and roxatidine on mucin biosynthesis of rat gastric oxyntic mucosa.

Although the involvement of nitric oxide (NO) in an increasing gastric mucus metabolism has been reported, information on whether or not its activation is limited to the specific mucus-producing cells is lacking. In this paper, we report the effect of the exogenous NO-donor, isosorbide dinitrate (ISDN), and second-generation histamine H2 receptor antagonist roxatidine (2-acetoxy-N-(3-[m-(1-piperidinylmethyl)phenoxy]propyl)acetamide hydrochloride) which is demonstrated to accelerate the mucin metabolism mediated by endogenous NO, on the mucin biosynthesis in distinct sites and layers of the rat gastric mucosa using an organ culture technique. Radiolabeled mucin was obtained from the tissue of full-thickness and the deep corpus layer, and the antrum of the rat stomach incubated for 5 hr with [3H]glucosamine(GlcN) in vitro. With the addition of ISDN to the culture medium, 3H-labeled mucin in the full-thickness corpus mucosa increased to 124-145% of the control (p<0.05), but not in the antrum. This stimulation of the mucin synthesis disappeared by the removal treatment of the surface mucous cell layer which has immunoreactivity of neuronal NO synthase. Similarly, roxatidine stimulated the mucin biosynthesis in the full-thickness corpus mucosa, but not in the gland mucous cell layer. These results suggest that the stimulation of the mucin biosynthesis mediated by NO is restricted to the surface mucous cells of the rat gastric oxyntic mucosa.     

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.     

Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP(3) in rat osteoblast-like cells

The existence of an osteotropic hormone (referred to as gastrocalcin) in the ECL cells of the gastric mucosa has been suggested. Both gastrin and an extract of the oxyntic mucosa lower blood Ca(2+) and stimulate Ca(2+) uptake into bone. The ECL cells are known to operate under gastrin control and, conceivably, gastrin lowers blood Ca(2+) indirectly by releasing the hypothetical ECL cell hormone. We have shown earlier that extracts of isolated ECL cells or of the granule/vesicle fraction of the oxyntic mucosa evoke a typical Ca(2+)-mediated second messenger response in osteoblastic cells. In the present study, we characterize this response further. An increase in intracellular inositol 1,4,5-trisphosphate (IP(3)) concentration was observed after treatment of UMR-106.01 osteoblast-like cells with extracts of ECL cells or granule/vesicle-enriched fractions from oxyntic mucosa. Intracellular cyclic adenosine monophosphate (cAMP) concentrations were not affected. Inhibition of phospholipase C (PLC) by U-73122 abolished the increase in [Ca(2+)](i). Preincubation of UMR-106.01 cells with pertussis toxin, which blocks many G-proteins, did not prevent the increases in IP(3) and [Ca(2+)](i). It was also found that the novel peptide hormone ghrelin, produced in the A-like cells of the oxyntic mucosa, did not evoke any Ca(2+) signal in osteoblastic cells. The results indicate that the extracts mediate their effects through a pertussis toxin-insensitive mechanism, and that binding to a receptor leads to activation of PLC and production of IP(3) resulting in increased [Ca(2+)](i). The putative osteotropic hormone is distinct from ghrelin.     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999     

Identification of glucagon-producing cells (A cells) in dog gastric mucosa

An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon.     

人正常腺上皮组织中MUC6基因的表达

应用免疫组织化学和原位杂交方法检测人正常腺上皮中MUC6基因的表达,揭示MUC6基因在正常人腺上皮组织中的分布异质性及其特点.结果显示MUC6基因编码的核心蛋白及其mRNA主要分布于正常胃粘膜胃腺的基底部,上皮细胞无MUC6基因表达,呈细颗粒状,位于细胞核周,胃底、胃窦的表达无区别;十二指肠绒毛上皮内的表达呈弥漫性,均质状,杯状和柱状细胞的表达类似,杯状细胞的粘液滴内未测得MUC6基因产物;空肠、结肠组织中无MUC6基因的表达;胆囊上皮组织内有强阳性MUC6核心蛋白的表达,而宫颈上皮中表达较弱.实验提示MUC6基因的表达存在异质性及器官特异性.     

Histamine in endocrine cells in the stomach. A survey of several species using a panel of histamine antibodies

Antibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions ("paracrine" appearance), running close to the base of the oxyntic-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a "paracrine" appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedgehog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man.(ABSTRACT TRUNCATED AT 400 WORDS)     

Histamine in endocrine cells in the stomach

Summary Antibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions (paracrine appearance), running close to the base of the oxyntico-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a paracrine appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedge-hog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man. In the oxyntic mucosa of the latter three species mast cells sometimes seemed to have an intraepithelial localization which made their distinction from endocrine cells difficult. In newborn cats (1–3 days old) in human foetuses (17–24 weeks gestational age) mast cells were relatively few in the gastric mucosa and the histamine-containing endocrine cells were easier to demonstrate as a consequence. Patients with achlorhydria (and pernicious anemia) or suffering from hypergastrinemia due to gastrinoma had a greatly increased number of histamine-storing endocrine cells in the oxyntic mucosa compared with normal individuals.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Alternating laminated array of two types of mucin in the human gastric surface mucous layer

Summary Attempts have been made to develop a procedure for preserving and analysing the surface mucous layer of the human stomach in paraffin sections. Histologically normal gastric mucosae were obtained from 20 surgically removed stomachs. Of the different fixatives tested, Carnoy's solution gave rise to the most satisfactory results. In Haematoxylin-Eosin stained sections, the surface mucous layer appeared as a thick eosinophilic layer coating the gastric mucosal surface and measured 55.4±2.5 m in the fundus and 21.8±1.0 m in the pylorus respectively. A dual staining method consisting of galactose oxidase-cold thionine Schiff and paradoxical concanavalin A staining was applied to the surface mucous layer in order to reveal the distribution pattern of mucins secreted by two types of mucous cell in the gastric mucosa: surface mucous cells and gland mucous cells. As a result of this staining, an alternating laminated layer was visualized which consisted of the particular two types of mucin. In five cases, the surface mucous layer was examined in unfixed frozen sections. This layer was only partially preserved but revealed the same laminated structure. These results indicated that gland mucous cell mucins contribute to form the surface mucous layer.     

Helicobacter pylori and the Surface Mucous Gel Layer of the Human Stomach

Background The colonization of Helicobacter pylori in the surface mucous gel layer (SMGL) was investigated.
Materials and Methods. Surgically removed stomachs were obtained from patients and included gastric ulcer (4 cases), duodenal ulcer (2), and gastric cancer (24). Five of these cases were examined at 8, 19, 28, 143, and 171 days after the end of eradication therapy. For the preservation of the SMGL, these specimens were fixed in cold Carnoy's solution, cleared in xylene, and embedded in paraffin. Serial sections were obtained and were stained by dual staining with the galactose oxidasecold thionin Schiff reaction followed by paradoxical Concanavalin A staining and immunostaining for H. pylori.
Results. H. pylori characterstically attached to surface mucous cells and colonized in the SMGL. H. pylori in the SMGL was more abundant than that attached to the surface mucous cells. The degree of H. pylori infection both on the surface of surface mucous cells and in the SMGL correlated well with the severity of gastritis. In the SMGL, this organism obviously preferred to colonize in the layer of surface mucous cell-type mucins, and the multilaminated structure of the SMGL deteriorated markedly. Eradication of H. pylori restored the structure of the SMGL, and the inflammatory reaction decreased gradually.
Conclusion. The SMGL is an indispensable site of H. pylori colonization, and this organism damaged the gastric mucosa partially by causing deterioration of the SMGL. Removal of the organism from the SMGL should be considered for eradication of this organism.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Identification of an apical Cl-/HCO3- exchanger in gastric surface mucous and duodenal villus cells

The molecular identity of the apical HCO3(-)-secreting transporter in gastric mucous cells remains unknown despite its essential role in preventing injury and ulcer by gastric acid. Here we report the identification of a Cl-/HCO3- exchanger that is located on apical membranes of gastric surface epithelial cells. RT-PCR studies of mouse gastrointestinal tract mRNAs demonstrated that this transporter, known as anion exchanger isoform 4 (AE4), is expressed in both stomach and duodenum. Northern blot analysis of RNA from purified stomach epithelial cells indicated that AE4 is expressed at higher levels in mucous cells than in parietal cells. Immunoblotting experiments identified AE4 as a approximately 110- to 120-kDa protein in membranes from stomach epithelium and apical membranes from duodenum. Immunocytochemical staining demonstrated that AE4 is expressed in apical membranes of surface cells in both mouse and rabbit stomach and duodenum. Functional studies in oocytes indicated that AE4 functions as a Cl-/HCO3- exchanger. These data show that AE4 is an apical Cl-/HCO3- exchanger in gastric mucous cells and duodenal villus cells. On the basis of its function and location, we propose that AE4 may play an important role in mucosal protection.     

Immunocytochemical localization of cathepsins B, H, and L in the rat gastro-duodenal mucosa

Cathepsins B, H, and L are representative cysteine proteinases in lysosomes of a large variety of cells. Previous immunochemical studies indicated the presence of these enzymes also in the gastrointestinal wall. Using specific antisera, the cellular and subcellular distribution of cathepsins B, H, and L in rat gastric (oxyntic and pyloric part) and duodenal mucosa was investigated by light and electron microscopical immunocytochemistry. The subtypes of cathepsins were distributed differently in the cellular constituents of the epithelia: Cathepsin B was localized to lysosomes of all cells except goblet cells. Cathepsin H was found predominantly in gastric parietal cells (lysosomes) and in secretion granules of pyloric gastrin and duodenal cholecystokinin cells. Cathepsin L immunoreactivities were weak and restricted to a minority of cells (gastric mucous cells, enterocytes). Interstitial cells of the lamina propria immunoreactive for cathepsins H and L were identified as macrophages. The present findings suggest a dual function of cathepsins in the gastro-duodenal mucosa. They (1) cleave enzymatically proteins and peptides ingested in lysosomes, and (2) they may be involved in the processing of biologically active peptides (enteric hormones) from their precursor proteins.     

Importance of acidic mucin secretions by foveolar and mucous neck cells of rat fundic mucosa as the defence mechanisms against HCl as revealed by fasting.

The localization of neutral mucin and acidic mucins in both control and fasted rat gastric fundic mucosa were examined by microscopic and electron microscopic histochemical methods. By Carnoy's fixation, the surface mucous coat of the control rat gastric fundic mucosa was found to be composed of alternating layers of acidic mucins and neutral mucin, indicating the synchronous and cyclic secretions of them. In many gastric pits of the fundic glands, the acidic mucins were found to spring out from the deep foveolar regions like volcanoes. This phenomenon may suggest that the acidic mucins play a fundamental role in protecting the pit cells against HCl during its passage, and the layers of neutral mucin and acidic mucins in the surface coat is the safeguard against the HCl and digestive enzymes in the gastric lumen. In the fasting rat gastric fundic mucosa, the acidity and the amount of the gastric juice were markedly decreased, indicating the suppressed secretions of mucins and HCl. The decreased production of sulfomucin was directly demonstrated by 35SO4-autoradiography. Many mucous neck cells existing in close association with the parietal cells were ballooned due to accumulation of alcian blue (AB)-positive but high iron-diamine (HID)-negative sialomucin, which was not demonstrable in the control. The secretory granules of sialomucin contained in the ballooned mucous neck cells were positively stained ultrastructurally with cacodylate-ferric colloid to stain acid mucopolysaccharides.