Histochemical reactions of gastrointestinal mucosubstances with orcein,high iron diamine and alcian blue after prior oxidation of tissue sections

Summary The histochemical orcein reaction (orc) for mucosubstances in tissue samples from the human gastrointestinal tract was compared with PAS, high iron diamine (HID) and Alcian blue reactions at pH 1.0 or 2.5 (AB 1 and AB 2.5). Orc, HID and AB 1 reactions were performed also with prior oxidation of the tissue sections with potassium permanganate or performic acid (ox-orc, ox-HID and ox-AB reactions, respectively). Orc reaction stained mucosubstances similarly to HID and AB 1; only the brush border and goblet cells in the colon were stained. The reactions of the mucosubstances obtained with ox-orc differed from those with PAS, HID, AB 1 or AB 2.5 but were similar to those with ox-HID or ox-AB; the mucosubstances in the brush border and the goblet cells in the colon and small bowel and in the foveolar epithelium of the stomach were strongly stained. Pyloric and cardiac glands were stained faintly with ox-orc but not with ox-HID or ox-AB. Brunner's glands were negative with ox-orc, ox-HID and ox-AB reactions. It was assumed that the orc reaction stains, like HID or AB 1, sulphate groups in epithelial mucosubstances, and that sulphonic acid residues, resulting from oxidation of disulphide groups in the protein core of mucus glycoproteins, are responsible for the ox-orc as well as for the ox-HID and ox-AB reactions.The study was supported by grants from the Cancer Society of Finland, Foundation of Orion Corporation and from the Paulo's Foundation, Helsinki, Finland     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections

Summary High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-HID and ox-HID) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive mucosubstances were found in the foveolar epithelium of the stomach and in goblet cells of small and large bowel.The study was supported by grants from Sigrid Juselius Foundation and Paulo Foundation, Helsinki, Finland     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections.

High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-Hid and ox-Hid) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive and in goblet cells of small and large bowel.     

结肠腺瘤的免疫组织化学及组织化学研究

本文应用免疫组织化学CEA、MC_5(PAP法)和组织化学AB(PH2.5)/PAS、HID/AB(PH2.5)染色方法,对43例结肠腺瘤、52例结肠腺瘤伴中、重度不典型增生,13例结肠腺瘤伴癌变,15例结肠腺癌和10例正常结肠组织进行了相关抗原及大肠粘蛋白的研究探讨。结果发现五类组织之间其着色含量都呈现一定的差异,尤其是随着腺瘤的异型程度增大,这种差异更为明显。本实验结果提示,上述染色法对结肠腺瘤癌变的逐渐演变过程的变化具有一定的参考价值。     

Histology and ultrastructure of the gut epithelium of the neotenic cave salamander, Proteus anguinus (Amphibia, Caudata)

Histological, histochemical, and ultrastructural features of the gut of the European endemic cave salamander Proteus anguinus were studied. The gut is a relatively undifferentiated muscular tube lined with a simple columnar epithelium containing numerous goblet cells. The mucosa and underlying lamina propria/submucosa are elevated into a number of high longitudinal folds projecting into the lumen. The enterocytes are covered apically with uniform microvilli. Irregularity in the arrangement of microvilli was observed. Occasionally, irregular protrusions of the cytoplasm appear between groups of microvilli. Pinocytotic activity occurs at the bases of the intermicrovillous space. Mitochondria are numerous in the apical cytoplasm and basally beneath the nuclei. The supranuclear cytoplasm contains most of the cell organelles. The lateral plasma membranes of adjacent cells interdigitate and are joined by junctional complexes. The periodic acid-Schiff (PAS) reaction, indicating neutral mucosubstances, is positive only in the apical brush border of enterocytes and in goblet cells. The goblet cells also stained with Alcian blue (AB), at pH 2.5, thus revealing the presence of carboxylated glycosaminoglycans. Compact aggregations of AB- and PAS-negative cells are situated directly below the epithelium. Mitotic figures are present in individual clusters of cells. The fine structure of cells in these clusters indicated that these cells could be responsible for renewal of intestinal epithelium. Numerous endocrine-like cells could also be seen. The closely packed smooth muscle cells and amorphous extracellular material with collagen fibrils constitute a net-like structure under the basal lamina that is very closely associated with the epithelium. There are numerous acidophilic granular cells between epithelial cells, in the lamina propria/submucosa, and between cells aggregations. They seem to be associated with nematode infections and possibly constitute a humoral defense mechanism.     

Comparative histochemistry of the mucoproteic cells of the hypophysis from Rhamdia hilarii, Hypostomus punctatus, Prochilodus scrofa and Cyprinus carpio (Teleostei). Immunohistochemical identification of the gonadotropic cells

Adenohypophyseal cells showing positive histochemical reactions for mucosubstances were classified as type I-IV in Hypostomus (Plecostomus) punctatus (Loricariidae), Rhamdia hilarii (Pimelolidae), Prochilodus scrofa (Prochilodontidae) and Cyprinus carpio (Cyprinidae) according to cell shape, size, cytological characteristics and adenohypophyseal distribution. Cell types I and II are common to the four species, with each cell type showing very similar cytological and histochemical characteristics, in spite of different adenohypophyseal distribution of cell type II, according to the teleost species. Type I cells are globular basophils located in the proximal pars ditalis and are positive to PAS and Alcian blue pH 2.5 (AB) reactions, showing cytoplasmic vacuoles and changes in granule concentration in the mature phase of the gonadal cycle. The smaller type II cells are fusiform or oval basophils exhibiting a strong AB reaction but also reacting to PAS. Type III cells are located in the pars intermedia showing PAS-positive reaction. Considering different teleost species, these cells exhibit some variations specially in relation to cell size and shape which are not detected in mature male C. carpio. Otherwise cell type IV is only present in the rostral pars distalis of P. scrofa. They are weakly basophilic and negative to PAS, reacting strongly to AB. Only cell type I showed unequivocally positive immunohistochemical results with anti-salmon gonadotropin.     

Histochemical and Biometric Study of the Gastrointestinal System of Hyla Orientalis (Bedriaga, 1890) (Anura,Hylidae)

This study was carried out to assess the localization of hyaluronic acid (HA) and the distribution of glycoproteins in the gastrointestinal system of adult Hyla orientalis. Histochemical analysis of the gastrointestinal system in H. orientalis showed that mucous content included glycogene and/or oxidable dioles [periodic acid/Schiff (PAS)+], neutral or acid-rich (PAS/AB pH 2.5+), sialic acid residues (KOH/PAS+) and acid sulphate [Aldehyde fuchsin (AF)+] glycoproteins. However the mucus content was not the same in stomach, small and large intestine. The mucus content of stomach included only glycogene and/or oxidable dioles and sialic acid residues. Besides these histochemical methods, the localization of HA was detected using biotinylated hyaluronic acid binding protein labeled with streptavidin-fluorescein isothiocyanate (FITC). In the extracellular matrix of the submucosa, the reaction for HA was evident. Since HA was located in submucosa beneath the epithelial layer of gastrointestinal system, it has a significant role in hydric balance, and essential to provide the gastrointestinal system integrity and functionality. According to biometric results, there were statistical differences between small and large intestine in terms of the amount of material stained positive with PAS/AB, PAS, KOH/PAS and AF/AB. Additionally, number of goblet cells in the small and large intestine was significantly different.Key words: Gastrointestinal system, goblet cell, glycoproteins, hyaluronic acid, amphibian, Hyla.     

Histochemistry of small intestinal dysplasia in familial polyposis coli

Biopsies of duodenal and ileal mucosa from patients with familial polyposis coli were studied. Areas of atypia were identified in the duodenum of six patients and in the ileum of three patients. Grade I atypia was characterized by crowding and elongation of cells and nuclei, a slight reduction in the number of goblet cells and the presence of a brush border; grade II atypia was further characterized by pseudo- or pluristratification of cells, a marked reduction in the number of goblet cells and the absence of a brush border. In areas of atypia, columnar cells often contained PAS-positive apical granules, which were diastase-resistant and unstained by alcian blue at any pH; the brush border, even where recognizable in haematoxylin-eosin and PAS-stained sections, was unreactive histochemically for alkaline phosphatase. Goblet cells were few in areas of atypia, but those present were regularly stained by PAS and alcian blue pH 2.6. Apical granules, similar in their histochemical characteristics to those observed in columnar cells in areas of atypia, were also found in otherwise normal mucosal areas, even in some patients with no overt areas of atypia in the biopsies studied. These granules have been interpreted as an abnormality, possibly preceding the onset of atypia. Hyperplasia of goblet cells, secreting mucins with the same staining pattern as in normal intestine, was found in some patients, either adjacent to areas of atypia or independent of them. Intervening columnar cells had a normal morphology, alkaline phosphatase-reactive brush borders and no sign of mucus secretion. This goblet cell hyperplasia has been interpreted as a reactive, nonspecific alteration of the mucosa.     

Mucosubstance histochemistry of Brunner's glands, pyloric glands and duodenal goblet cells in the ferret

The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods. The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man. The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomucins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Tunicate Endostyles: Histochemistry of Acidic Glycoproteins Re-examined in Ciona intestinalis and Styela plicata

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones. The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride. Polyanions were stained with alcian blue (AB) at pH 2.5 or azure A, while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AH) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 1.0 responses. Almost invariably zone 6 was AB+ at pH 2.5; zones 2 and 4 were frequently responsive to AB, but less intense. Each of these 3 zones, when AB+, was sensitive to AH. Responses by zones 3 and 5 to AB (pH 2.5), azure A and saponification suggest that these zones contain mostly nuclear material. In secretory zones 2, 4 and 6 histological responses are consistent with the histochemistry of sialomucins. Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for Ciona, only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains.     

南方鲇消化道杯状细胞分布及类型探讨

本研究应用阿利新蓝－过碘酸雪夫氏（ＡＢ－ＰＡＳ）染色方法对南方鲇（Ｓｉｌｕｒｕｓ　ｍｅｒｉｄｉｏｎａｌｉｓ）消化道杯状细胞的分布特点进行了分类研究,结果表明：南方鲇消化道杯状细胞的数量从肠前段、肠中段到肠后段,呈现递增趋势,有杯状、囊状和梨形三种不同的形态,ＡＢ反应阳性,ＰＡＳ反应阴性,分泌物主要含酸性粘多糖,属Ⅱ型粘液细胞。     

Alcianblue/PAS or PAS/alcianblue?

Summary The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-posltive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Mucosubstance histochemistry of Brunner's glands,pyloric glands and duodenal goblet cells in the ferret

Summary The mucosubstance of Brunner's glands, pyloric glands and duodenal goblet cells were studied using the various histochemical methods.The secretions of both Brunner's and pyloric glands were similar in their histochemical reactions. They contained neutral mucosubstances as in these glands in man.The duodenal goblet cells showed variations in their histochemical characters. (i) The secretions of most of the deep cells and the majority of superficial cells contained sialidase-labile and sialidase-resistant sialomucins. (ii) There were a few superficial and occasional deep cells, the secretions of which contained sulphated mucosubstances. (iii) There were some goblet cells, more in the villi than in the crypts, the secretions of which contained a mixture of sialomycins and a sulphated mucin. The sialomucin was mostly sialidase-labile and partly sialidase-resistant.     

Alcianblue/PAS or PAS/alcianblue? Remarks on a classical technic used in carbohydrate histochemistry

The combined alcian blue (AB)/PAS technic is widely used for the detection and characterization of mucosubstances in tissue sections. Mostly the sequence AB/PAS is used, occasionally also the reserved sequence PAS/AB. The present study shows clearly that the sequence of the combined technic, i.e. AB/PAS or PAS/AB is substantially influencing the results. So it could be demonstrated that by using the combination PAS/AB originally PAS-positive and AB-negative reacting mucosubstances become AB-positive. This could be caused by periodic acid oxidation followed by addition of hydrogen sulfite to aldehyde group thus providing secondary basophilic resp. AB positive material.     

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.     

Influence of sulphate groups in the binding of peanut agglutinin. Histochemical demonstration with ligh- and electron-microscopy

Summary The influence of sulphation of mucus glycoproteins in the binding of peanut agglutinin (PNA) to tissue sections has been investigated by means of histochemical techniques at the light- and electron-microscopic level. A sequential methylation-saponification procedure was applied for the desulphation of tissue samples. Labelling by peroxidase- and colloidal gold-conjugated PNA was compared in control and desulphated samples of rat intestinal mucosa. The high-iron-diamine (HID) technique was used as a control for the effectiveness of the desulphation technique, and the Alcian Blue, pH 2.5 (AB 2.5), PAS and phosphotungstic acid-HCl (acid-PTA) techniques served as controls for the integrity of the oligosaccharide chains, respectively. In general, a marked increase of PNA reactivity was observed in desulphated samples when compared with control sections. These findings indicate that sulphation of galactose inhibits the binding of PNA to carbohydrate moieties in tissue sections. Staining patterns obtained with HID, PNA and the desulphation-PNA sequence in the goblet cells of the large intestine suggest a modification of the secretory product stored in these cells as the cell matures and moves from the lower crypt region toward the luminal surface. These modifications were not detected in the small intestine. Ultrastructural detection of PNA-binding sites suggests that galactose residues are incorporated into the oligosaccharide chains of O-liked glycoproteins at the medial cisternae of the Golgi apparatus. However, sulphation occurs at the trans side of the Golgi complex and the trans Golgi network. In conclusion, desulphation procedures are useful for revealing PNA-binding sites.     

The fine structure of the stomach mucosa of the llama (Llama guanacoe)

Summary The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron density of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.     

Organogenesis of the digestive tract in the white seabream,Diplodus sargus. Histological and histochemical approaches

The ontogeny of the digestive tract of the white seabream, Diplodus sargus during the larval development up to day 45 post-hatching (dph) has been studied using histological and histochemical techniques. The oesophageal goblet cells appeared around 6 dph and contained neutral and acid mucosubstances (PAS/diastase-PAS and Alcian Blue pH 2.5 positive reactions). An incipient stomach can be distinguished from 2 dph but the first sign of gastric gland development was detected around 13-15 dph, increasing in number and size by 22-23 dph. Gastric glands were concentrated in the cardiac stomach region and they had a high content of protein rich in tyrosine, arginine and tryptophan. Acidophilic supranuclear inclusions related to pynocitosis of proteins, were already observed in the intestinal cells of the posterior intestine around 4-6 dph (exogenous feeding) and they were present until 25 dph. The intestinal mucous cells appeared between 15-18 dph and contained a mixture of neutral and acid mucosubstances/glycoconjugates, carboxylated ones being more abundant than the sulphated ones. The stomach and gastric glands were fully developed by the first month of life marking the beginning of digestive features characteristic of the juvenile stage. Around 4-6 dph, glycogen, proteins and neutral lipids were observed in the granular cytoplasm of hepatocytes. Strongly acidophilic zymogen granules were also present, at this time, in the basophilic cytoplasm of the exocrine pancreatic acinar cells and contained abundant proteins, especially rich in arginine, tyrosine and tryptophan.     

小肠上皮分泌细胞特性的组织学观察

目的寻找一种可以替代人体消化管的动物标本,并通过特殊染色方法,使得小肠上皮分泌细胞的形态特征能够明显地显示出来。方法随机采集成年猫小肠的新鲜标本,经Bouin液灌注固定24h后,石蜡包埋切片脱蜡入水。分别采用Gomori染色法、PAS反应、Gomori＋PAS反应、阿利新蓝（alcian blue,AB）染色法、AB＋PAS反应、HE染色法和苏木精-焰红染色法进行染色。结果在各种染色的切片标本上,能够观察到杯状细胞的形态、分布和染色特性以及肠内分泌细胞的特点,并发现在它们之间还存在一种绿色颗粒细胞和嗜酸性颗粒细胞。结论通过特殊染色可以肯定猫的小肠杯状细胞合成的是中性粘蛋白和酸性粘蛋白;绿色颗粒细胞为未成熟杯状细胞;嗜酸性颗粒细胞为Paneth细胞,其特点是单个分散分布。肠内分泌细胞与周围其他上皮细胞的染色对比明显而容易识别。     

Analysis on origin recognition complex containing Orc5p with defective Walker A motif

Orc5p is one of six proteins that make up the origin recognition complex (ORC), a candidate initiator of chromosomal DNA replication in eukaryotes. To investigate the role of ATP binding to Orc5p in cells, we constructed orc5-A, a strain of Saccharomyces cerevisiae having a mutation in the Walker A motif of Orc5p (K43E). The strain showed temperature-sensitive growth. Incubation at a nonpermissive temperature (37 degrees C) caused accumulation of cells with nearly 2C DNA content. Overproduction of Orc4p, another subunit of ORC, suppresses this temperature sensitivity, but overproduction of other subunits did not. Overproduction of Orc4p did not suppress the temperature sensitivity of another orc5 mutant, orc5-1, whose mutation, L331P, is outside the ATP-binding motif. These results suggest that Orc4p is specifically involved in ATP binding to Orc5p itself or its function in DNA replication. Immunoblotting experiments revealed that in the orc5-A strain at a nonpermissive temperature, all ORC subunits gradually disappeared, suggesting that ORC5-A becomes degraded at nonpermissive temperatures. We therefore consider that ATP binding to Orc5p is involved in efficient ORC formation and that Orc4p is involved in this process.