Low Iron Diamine Research Articles

Oregano Feed Supplementation Affects Glycoconjugates Production in Swine Gut.

Simple SummaryThe European ban towards antibiotics is increasing the number of studies on the effects of feed additives, such as plant extracts, in order to enhance the health and welfare status of domestic animals intended for human consumption. Origanum vulgare possesses multiple pharmacological characteristics and its antioxidant and antibacterial properties are particularly interesting. Besides, a recent study aimed at evaluating the effects of oregano aqueous extract supplementation in poultry nutrition gave encouraging results regarding the secretion of glycoconjugates in the gut which increases tissue hydration and protects the intestinal mucosa from pathogenic bacteria, viruses, and parasites. Therefore, we investigated the effects of oregano feed supplementation on antioxidant and defense ability of pig gut. Our results showed that there was improved production of glycoconjugates in the duodenum and colon of pigs fed with supplementation of oregano aqueous extract, enhancing protection of the mucosa of these sections of the intestine. Also, we observed an enhanced antioxidant action in the two examined gut tract samples of the group supplemented with oregano. Findings can be used in further research to identify ways to improve endogenous defense ability with to reduce antibiotic use and prevent antimicrobial resistance.This study evaluated the effects of adding oregano aqueous extract (OAE) to the diet of pig slaughtered at finisher stage. Study was performed to identify glycoconjugates and evaluate the oxidative stress levels in the duodenum and colon intestinal tracts. Glycohistochemistry was performed by staining with Periodic acid–Schiff (PAS), Alcian blue (AB) pH 2.5, AB-PAS, AB pH 1, AB pH 0.5, low iron diamine, and high iron diamine. Serial sections were pre-treated with sialidase V before staining with AB pH 2.5 (Sial-AB) preceded or not by saponification. To study oxidative stress, an immunohistochemical analysis was applied to investigate the presence of the oxidative stress target molecule Bcl-2 Associate X protein (BAX). Findings show that oregano aqueous extract supplementation improves the production of the secretion glycoconjugates involved in direct and indirect defense, thus enhancing the protection of the pig intestinal mucosa. Moreover, the reduced BAX protein immunostaining observed in both duodenum and colon of swine of the oregano-supplemented group respect to that observed in the control group suggests an enhanced antioxidant action by oregano adding. Findings could be useful for other studies aiming to reduce antibiotic use and prevent antimicrobial resistance.

Glycocomposition of the apocrine interdigital gland secretions in the fallow deer ( Dama dama)

The secretions of the tubular interdigital glands were investigated by conventional (Periodic-Acid Schiff, Alcian-Blue at different pH, Low Iron Diamine and High Iron Diamine) and lectin (Con-A, UEA-I, LTA, WGA, GSA-II, GSA-IB4, SBA, PNA, ECA, DBA, MAL-II and SNA) histochemical methods in adult males and females of different age of fallow deer during the breeding season. Sialidase digestion and deacetylation pre-treatment were also employed in conjunction with lectin histochemistry. The glandular epithelium consisted of a single layer of low columnar cells with typical apical protrusions. No substantial differences of the above histochemical staining in relation to sex and age were observed. Conventional histochemical staining revealed that the interdigital glands secreted neutral glycoproteins whereas acidic glycocomponents did not seem to be present. Lectin histochemical technique allowed us to disclose a great heterogeneity of glycoproteins with N- and O-linked oligosaccharides containing α- d-Man/α- d-Glc, GlcNAc, α-Fuc, terminal β- d-Gal-(1-3)- d-GalNAc, - d-Gal-(1-4)- d-GlcNAc, α-Gal and β-GalNAc residues. β-GalNAc and disaccharide β- d-Gal-(1-3)- d-GalNAc were also found as subterminal to sialyl moieties. The lack of sexual and age-related differences in the glucidic content of the glandular secretions seems to indicate that the glycoderivatives may play only an accessory role in the production of odoriferous signals in fallow deer.

Complex carbohydrates occurring in the digestive apparatus of Umbrina cirrosa (L.) fry.

The glycosoaminoglycans in the digestive apparatus of immature fish have important biological functions and are involved in morphofunctional differentiation. The aim of this study was to investigate the glycoconjugate histochemistry in the different parts of the digestive apparatus (oesophagus, stomach, intestine) of Umbrina cirrosa (L.) fry using classical histochemical reactions (periodic acid-Schiff, Alcian blue pH 2.5, Alcian blue/periodic acid-Schiff, high iron diamine, low iron diamine) in conjunction with glycolytic digestions that degrade different classes of glycosoaminoglycans. No differences were observed in the reactivity to conventional histochemical staining of the oesophagus, stomach or intestine among 27-, 34- or 44-day-old fry. In the oesophagus, the mucopolysaccharides contained chondroitin sulphates B and A and/or C, heparan sulphate and chondroitin. In the stomach, only neutral glycoconjugates were revealed, whereas in the intestine there were only chondroitin sulphates. Some differences in the type and content of glycoconjugates were found in Umbrina cirrosa (L.) fry compared to those of adult subjects, probably related to different dietary habits and to changes in the environmental conditions.

Histochemical detection of the lectin-binding carbohydrates in the zona pellucida during oocyte growth in the wild boar (Sus scrofa scrofa).

The changes that occur in the carbohydrate composition of zona pellucida glycoproteins during oocyte maturation in the wild-boar were studied using periodic-acid Schiff (PAS), High Iron Diamine (HID) and Low Iron Diamine (LID). Lectin staining was performed with a panel of 11 HRP-lectin conjugates combined with neuraminidase digestion and chemical treatments. There were few internal glucidic residues, such as N-acetylglucosamine, in the wild boar zona pellucida but there were many subterminal beta-N-acetylgalactosamine, alpha- and beta-galactose determinants masked by sialic acid. In addition, beta-N-acetylgalactosamine, beta-galactose-(1-3)-N-acetylgalactosamine and beta-galactose-(1-4)-N-acetylglucosamine were detected in the sulphated form in the terminal and/or subterminal position. Some differences in the lectin reactive sites occurred in the zona pellucida, depending on the stage of oocyte maturation.

Histochemical analyses of glycosaminoglycans in the synovial membrane of the canine knee joint

The accurate localization and nature of glycosaminoglycans in the synovial membrane of the canine knee joint were examined histochemically by means of the selective sensitized diamine procedures based upon high and low iron diamine stainings in combination with enzyme digestions. Using these methods, it was possible to clearly and easily detect exceedingly small amounts of glycosaminoglycans in synovial tissues, which cannot be visualized by methods employed to date. The sensitized high iron diamine (S-HID) procedure resulted in positive reactions of varying intensities in the intercellular matrix of synovial intima, and in the extracellular matrix and small capillary walls of the superficial layer in the synovial subintima, and also reacted vividly in the extracellular matrix and blood vessel walls of the deeper layer in the synovial subintima. In particular, the sensitized low iron diamine (S-LID) procedure resulted in positive reactions of the extracellular matrix in the synovial subintimal layers. The S-HID and S-LID procedures combined with the enzyme digestions proved that glycosaminoglycan molecular species such as chondroitin sulfate A/C, dermatan sulfate, heparan sulfate and hyaluronic acid are present in various concentrations in the synovial membrane of the canine knee joint. The present results were discussed with reference to the histophysiological and pathophysiological functions of glycosaminoglycans in the synovium of domestic mammals.

Histochemical Studies of Glycosaminoglycans in the Developing Eyelids of Experimental Microphthalmic ( Cts) Mice

Purpose: The Cts mouse is a mutant strain which induces congenital cataract and small eyes in homozygotes. The process of eyelid development in Cts mice was examined histochemically. Materials and Methods: Prenatal (from day 14 of gestation) and postnatal (to day 14) animals of unaffected and homozygous mice were examined. Sensitized high iron diamine staining was done after chondroitinase B/testicular hyaluronidase double digestion, and sensitized low iron diamine staining was done after Streptomyces hyaluronidase digestion. Results: The main chondroitin sulfate isomer in the dermis of the eyelid changed from chondroitin sulfate A/C to chondroitin sulfate B at birth in the unaffected mice. In the homozygotes, the same change took place 4 days after birth. In the dermis of the eyelid, hyaluronic acid was first detected on day 14 of gestation and gradually increased until birth in both unaffected and homozygous mice. There was slightly more hyaluronic acid in homozygous mice until 4 days after birth. Conclusion: The present study showed that the maturation of glycosaminoglycan molecular species in the eyelid was retarded in Cts homozygotes. (J Jpn Ophthalmol Soc 103:425–431, 1999)

Glycohistochemical investigation of canine and feline zonae pellucidae of preantral and antral oocytes

Glycoconjugate modifications were analysed in the zona pellucida during development of oocytes in dog and cat using conventional histochemical staining methods with or without previous carbohydrate digestion. A series of lectins combined with desulphation and sialic acid degradation were applied. No differences were observed between dog and cat follicles using conventional histochemical staining methods. In both species, the zona pellucida and follicular fluid/intercellular matrix strongly reacted with PAS and high iron diamine stain (HID) and reacted moderately with low iron diamine stain (LID). Treatment with testicular hyaluronidase, chondroitinase ABC, chondroitinase AC and chondroitinase B treatment diminished HID and LID positivity of follicular fluid and intercellular matrix. Lectins that gave the most intense staining of the zona pellucida of both species were SBA, PNA, RCA-I, GSA-IB4 and WGA, indicating the presence of beta-D-GalNAc, D-Gal and GlcNAc residues. Sulpho- and asulpho-carbohydrates were identified in terminal and/or subterminal positions linked to sialic acid residues. In conclusion, the results indicate that glycosaminoglycans are not present in the zona pellucida of both species. Differences were observed in carbohydrate residues and in their spatial distribution, depending on species and developmental stage of the follicles. The similarity in lectin affinity between ooplasm and zona pellucida of oocytes present in follicles at different stages of development confirm the involvement of oocytes in zona pellucida production.

Characterization of the complex carbohydrates in the zona pellucida of mammalian oocytes using lectin histochemistry.

The objective of this study was to characterize the glycoconjugates present in the zona pellucida of the follicular oocytes in sheep, goats and pigs. The zona pellucida was stained with periodic acid-Schiff, low iron diamine, high iron diamine, and nine different lectin horseradish conjugates: Con-A, SBA, DBA, PNA, RCA-I, GSA-II, WGA, LTA and UEA-I. Staining with DBA, PNA, SBA and RCA-I was performed with and without saponification with KOH and sialidase digestion. The results showed the presence of neutral and acidic glycoconjugates with different terminal sugars and also sialic acid radicals in the zona pellucida of all the animal studied. In particular, the positive staining with WGA, SBA, PNA and RCA-I suggests the presence of oligosaccharides with N-acetyl-D-glucosamine and sialic acid linked to the penultimate beta-N-acetyl-D-galactosamine and to the disaccharide galactosyl-(beta 1-3)-N-acetyl-D-galactosamine. The terminal trisaccharide sialic acid galactosyl-(beta 1-4)-N-acetyl-D-glucosamine was identified only in the zona pellucida of ovine and porcine oocytes. Thus, the zona pellucida exhibited species-specific variations in the content and distribution of lectin-binding patterns that may reflect the species specificity of gamete interaction.

Ultracytochemistry of glycoconjugates in pig duodenal gland

To elucidate the ultrastructure, glycoprotein profile and site of glycosylation of glandular cells in relation to the functional polarity of the organelles, swine duodenal tissue was embedded in glycol methacrylate and subsequently stained for periodic acid thiocarbohydrazide silver proteinate (PA-TCH-SP), high iron diamine (HID)-TCH-SP, low iron diamine (LID)-TCH-SP, ninhydrin T-TCH-SP and five peroxidase-labeled lectins. The secretory granules in the duodenal gland cells were electron lucent with a 200 nm to 500 nm electrondense core. Glycoconjugates were confined to the secretory granules and elements of the Golgi complex. Protein activity was located only in the electron dense core. Achivementestic staining pattern for Concanavalin A (Con A), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) was observed in stained secretory granules and the Golgi apparatus. A few cis cisternae were stained with SBA and UEA-I. Trans cisternae were stained with WGA and PNA. Con A reacted with seromucous granules and rough endoplasmic reticulum. These observational findings suggest that these are seromucous cells. The Golgi apparatus is the site of glycosylation and can be divided into two distinct compartments.

Standardization of staining in glycosaminoglycan histochemistry: alcian blue, its analogues, and diamine methods.

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results in particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables histochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.

Histochemistry of carbohydrates as performed by physical development procedures.

The histochemistry of carbohydrates demonstrated by means of physical development procedures has been reviewed in terms of the use and reliability of the procedures, physical developers, practice of the procedures, a fundamental series of light and electron microscopic methods and certain other promising aspects of this area of histochemistry. A line of fundamental light- and electron-microscopic histochemical methods for carbohydrates using physical development procedures such as periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD), high- or low-iron diamine (HID or LID)-TCH-SP-PD and lectin-gold (LT-G)-PD and related methods has been found to be more efficient, compared with those without physical development procedures. Since a series of other promising histochemical methods for carbohydrates using physical development procedures have been derived or are now being introduced, these procedures could be regarded as an unusually potent vehicle for effectively advancing carbohydrate histochemistry in both light and electron microscopy.

Schistosoma mansoni: The chemical nature of the secretions produced by the Mehlis' gland and ootype as revealed by cytochemical studies

The ‘secretory pathway’ of the Mehlis' gland secretion as well as the maturation of the secretory granules are described. The secretions produced by the Mehlis' gland and the distal ootype in Schistosoma mansoni were identified as neutral glycoproteins. They were both periodate reactive, stained with phosphotungstic acid at low pH, and sensitive to the proteolytic action of papain. The secretions did not strain with both the low-iron diamine and the high-iron diamine methods for the presence of acidic glycoconjugates, and they did not contain a detectable level of sulphydryl groups. The possible role of the secretion from the Mehlis' gland in egg-shell formation is discussed.

Ultrastructural localization of secretory glycoconjugates in the gallbladder epithelium of the Richardson’s ground squirrel before and during cholesterol gallstone formation

Hypersecretion of mucin is a critical feature of gallbladder epithelial cells in ground squirrels fed a 2% cholesterol-enriched diet for the induction of cholesterol gallstone formation. The timing of this physiological response indicates a potential role for mucin as an initiator of cholesterol crystal nucleation from supersaturated bile. There is little information regarding the specific type of glycoprotein secreted by gallbladder epithelia and routine light microscope histochemistry proved unproductive for detection of specific glycoprotein types being synthesized as the intracellular accumulations were not large enough to permit visualization of a colored precipitate. Ultrastructural histochemistry was utilized to differentiate between the two main mucin types secreted by the gastrointestinal tract - sulphated and sialomucins.Two ultrastructural methods utilizing the histochemical basis of the high and low iron diamine techniques were used. The high iron diamine (HID) technique imparts density to sulphated complex carbohydrates while the low iron diamine (LID) method imparts density to acidic glycoconjugates.

Diamine-thiocarbohydrazide-silver proteinate- physical development methods for the histochemical detection of acidic glycoconjugates in electron microscopy.

A couple of new sensitive methods have been established for the histochemical detection of acidic glycoconjugates in electron microscopy. These are combined staining methods consisting of a high or low iron diamine preembedding and thiocarbohydrazide silver proteinate postembedding sequence (HID or LID-TCH-SP) followed by a physical development (PD) procedure. The HID or LID-TCH-SP-PD methods have been applied to a series of acidic glycoconjugate-containing ultrastructures from different organs of the rat and mouse such as the aorta, kidney, pancreas and spleen, in comparision with the HID or LID-TCH-SP techniques. The new methods are signigicantly more sensitive than the HID or LID-TCH-SP technique. Their specificity is sufficient.

Carbohydrate cytochemistry of rhesus monkey tracheal submucosal glands

This study was designed to characterize the ultrastructure and carbohydrate content of secretory cells in submucosal glands of rhesus monkey and to compare this information with that available for humans. The tracheas from five adult monkeys were fixed by airway infusion, processed, and embedded for both light and transmission electron microscopy. Histochemical strains including alcian blue-periodic acid-Schiff, dialyzed iron, and high-iron diamine-alcian blue were applied to serial glycol methacrylate sections. The cytochemical stains used included periodic acid-thiocarbohydrazide-silver proteinate, high-iron diamine, and low-iron diamine. The glandular secretory cells were divided into four categories based on ultrastructure and location within the gland. Cells in the first category resembled the mucous cell of the surface epithelium and were located in ducts most proximal to the tracheal lumen. The second category consisted of cells that were located in distal ducts and contained large electron-lucent granules. The granules in both of these cell groups contained material that was periodate-reactive and sulfated. Cells of the third category contained granules that were either electron-lucent or electron-dense. These cells, which were difficult to characterize as either serous or mucous, were located in secretory tubules and acini and contained periodate-reactive glycoconjugates that were either sulfated or nonsulfated. The last category consisted mainly of cells that contained electron-dense granules that were lightly periodate-reactive or a few that were unreactive with any of the cytochemical methods used here.(ABSTRACT TRUNCATED AT 250 WORDS)

The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica.

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.

Fine localization of sulphated and non-sulphated glycoconjugates in the rabbit oviduct during the estrous cycle

An ultrastructural histochemical investigation was carried out to ascertain the localization of the glucidie components and to verify the histochemical modifications during the physiological estrogenic cycle of rabbit oviduct. Stainings were performed with high iron diamine (HID), low iron diamine (LID), dialized iron (DI), tannic acid-uranyl acetate (TA-U), and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) methods. The findings indicated that the secretory activity is more intense in the isthmus than in the ampulla. Neutral glycoconjugates are localized both in the secreting elements of the ampulla and in those of the isthmus, while carboxylated and sulphated glycosaminoglycans are localized only in the secretory granules of the secreting elements of the isthmus.

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues.

The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosamino-glycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.

Carbohydrate cytochemistry on hypodermic lymphatic endothelium of the green lizard, Lacerta hispanica.

A cytochemical study on the endothelium of the hypodermic lymphatic capillaries of the green lizard, Lacerta hispanica, has been carried out. The dialysed iron method produced a homogeneous precipitate on the surface of the endothelial cells and on the inside of the endocytic vesicles. The periodic acid-thiocarbohydrazide-silver proteinate, low pH phosphotungstic acid and high iron diamine techniques gave negative results. The carbohydrates in the capillaries thus seem to be glycosaminoglycans with carboxyl groups. The possible role of these glycosaminoglycans in the formation of the endocytic vesicles is discussed.

Ultrastructural visualization of complex carbohydrates in eosinophilic leukocytes.

Complex carbohydrates in eosinophils from human, rabbit, and rat marrow were identified and localized by cytochemical and radioautographic methods. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP), low iron diamine (LID)-TCH-SP, and periodate (PA)-TCH-SP methods were used for the localization of sulfate, sulfate and carboxyl, and vicinal glycol- containing complex carbohydrates, respectively. Golgi vesicles and small precursor granules (0.2-0.4 micrometer in diameter) demonstrated strong HID-TCH-SP staining and labeled intensely after a 10-minute pulse with 35SO4(2). Crystalloid-free or immature specific granules (0.5-0.9 micrometers in diameter) labeled heavily after a 60-minute incubation and 60-minute chase with 35SO4(2). Immature granules were graded according to their HID-TCH-SP staining. Type I granules demonstrated strong rim staining and similar or somewhat less central staining, whereas type 2 granules only demonstrated rim staining, and type 3 granules lacked staining. Fully mature crystalloid-containing granules lacked staining. LID-TCH-SP similarly stained the HID-positive sulfated material in cytoplasmic granules. PA-TCH-SP stained some Golgi vesicles and diffusely stained all precursor granules and type 1 granules. Weaker staining was observed in type 2 granules and staining was very weak or absent in type 3 and crystalloid-containing granules. In early eosinophils, tubulovesicular structures (TVS) were observed rosetting and contacting precursor and type 1 granules. These TVS contained material with strong PA-TCH-SP staining but lacked HID-TCH-SP or LID-TCH-SP-reactive acidic glycoconjugates. Flattened Golgi saccules or early eosinophils stained weakly or not at all with the PA-TCH-SP method. Small granules and TVS in late (bilobed) eosinophils displayed PA-TCH-SP reactivity and lacked HID-TCH-SP staining but differed from TVS in early eosinophils in that they were not associated as rosettes with specific granules. These results indicate that sulfated and vicinal glycol-containing complex carbohydrates are differently disturbed in immature specific granules of eosinophils and presumably become masked to staining as the granule matures.