Proteolysis and lectin histochemistry

Summary Lectin binding to formalin-fixed, paraffin-embedded tissue can often be enhanced by pre-treatment of the sections with proteolytic enzymes. However, the pattern of staining may be profoundly influenced by the type of enzyme preparation which is used.Sites of binding of thirteen different lectins to murine ovary and thyroid gland were studied after exposure of tissue sections to crude trypsin, purified trypsin, purified -chymotrypsin, pepsin, protease VII, papain, bromelain, thermolysin or elastase. With most lectins, the results obtained were similar regardless of which enzyme was used for proteolytic digestion. However, the pattern of binding of soy bean lectin to the ovary and of concanavalin A and common pea lectin to the thyroid gland was highly dependent upon the enzyme used to pre-treat the sections. In both tissues, the staining pattern seen in untreated frozen sections was similar to that found in formalin-fixed, paraffinembedded material digested with purified trypsin, but was different from that observed after exposure of processed sections to crude trypsin. The location of binding sites after treatment of paraffin sections with chymotrypsin was the same as that after digestion with crude trypsin. Results obtained after the use of other proteolytic enzymes varied according to the tissue being studied.These findings imply that the effect of treatment with crude trypsin is due to contaminating chymotrypsin, and demonstrate that the use of purified trypsin may have advantages over other proteolytic enzymes in lectin histochemistry. The observations may also apply to other related cytochemical techniques such as immunocytochemistry.     

Resolution of glycoproteins by a lectin gel-shift assay

Gel-shift assays previously described in the literature are based on protein-protein or protein-DNA interactions. We show that carbohydrate-lectin interactions can be successfully used to alter the electrophoretic mobility of glycosylated, but not nonglycosylated, protein species in SDS-polyacrylamide gels. We were able to separate the two closely migrating mono- (95 kDa) and nonglycosylated (92 kDa) forms of a polytopic membrane protein, anion exchanger 1 (AE1), synthesized by cell-free translation or in transfected HEK293 cells. Concanavalin A was selected as the lectin due to the high mannose content of the oligosaccharide chain on AE1. Concanavalin A was either added to the samples prior to loading or copolymerized in a top layer of the separating gel, the latter being the method of choice. The presence of concanavalin A resulted in slower mobility of the monoglycosylated protein while the mobility of the nonglycosylated form was not altered. The shift in mobility was dependent on concentration of concanavalin A and the length of separating gel containing copolymerized concanavalin A. When a diglycosylated mutant of AE1 was tested, good separation was achieved at lower concentrations of concanavalin A. This lectin gel-shift assay allows the separation of N-glycosylated and nonglycosylated forms of the protein.     

Lectin histochemistry of feline sphingomyelinosis.

The brain from a Siamese cat with sphingomyelinosis was examined with lectin histochemistry. Swollen neurons were stained with Canavalia ensiformis agglutinin (Con A). Some of them were also stained with Ricinus communis agglutinin-I (RCA-I) and Ulex europaeus agglutinin-I (UEA-I). A small number of axonal spheroids and glia cells were positive for Con A, RCA-I, UEA-I and wheat germ agglutinin. Control tissues were weakly stained with Con A, but not with any of the other lectins. These results indicate that affected neurons contain mannose and glucose residues in addition to sphingomyelin. This study points to the possibility that the characteristics of lectin histochemical study might be helpful for the diagnosis of sphingomyelinosis.     

Demonstration of endocytosis vesicles by means of electron-microscopic histochemistry

Summary A phosphatase activity similar to that of the plasma membrane is demonstrated at the inside of the membrane, which delimits the lipid-containing lysosomes of chicken liver parenchymal cells. This is taken as a definite confirmation of their endocytotic origin.It should be possible to demonstrate endocytosis vesicles in other cells in this way. It is, however, pointed out, that cells with a weak plasma membrane activity may require particular attention to certain steps in the technical procedure.This study was supported in part by a grant from the Swedish Medical Research Council.     

Pregnancy-related changes in the human endometrium revealed by lectin histochemistry

The binding of 22 fluorescein isothiocyanate (FITC) conjugated lectins to human proliferative phase and pregnant endometrium was studied histochemically. Only the lectin from Bauhinia purpurea (BPA) reacted exclusively with the epithelial cells. All the others reacted to a certain extent with glandular and/or stromal cells. Lectins from soybean (SBA), and Vicia villosa seeds (VVA) reacted with endometrial glands of pregnancy but not with the glands of the proliferative endometrium. In the proliferative endometrium SBA reacted only with cells of the surface endometrium. Lectin from peanuts (PNA) reacted only with some glands in the proliferative endometrium but was unreactive with others. In pregnant endometrium PNA reacted with all glands. Lectins from lentils (LCA) and red kidney beans (PHA-E and PHA-L) reacted with endometrial glands of the proliferative phase but not with the glands from pregnant endometrium. We thus show that FITC labeled lectins define specific carbohydrate moieties selectively expressed on either proliferative phase or pregnant endometrial glands.     

Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry

Knowledge of chemical structure of glycoconjugates (GCs) at precise loci has increased through histochemical use of a battery of horseradish peroxidase-conjugated lectins, each possessing affinity for a specific terminal sugar or internal sugar linkage. Lectin histochemistry has shown tremendous variability among GCs in different histologic sites, reflecting known chemical diversity of these substances. GCs differ in structure among various cell types in an animal and differ at a given histologic site between species or between individuals in outbred but not inbred species. Lectin conjugates react with and detect GCs not otherwise demonstrable histochemically and, because of low concentration in tissue, not identified biochemically. Lectin-HRP conjugates have visualized unique GC with terminal GalNAc in primordial germ cells of rat embryos, with terminal Gal in epithelial basal cells of rodents and nodes of Ranvier in rats and with terminal GalNAc in a cell population in the thymus, Peyer's patches and intestinal lamina propria of some but not other mice.     

Characterization of glycoconjugates in the bovine endometrium and chorion by lectin histochemistry

Lectin binding patterns were examined on normal bovine endometrium and bovine placentomes during four stages of pregnancy using 13 biotinylated lectins. Lectin binding intensity increased in early pregnancy for many lectins, whereas binding to fucosyl residues decreased. Persistence of strong lectin binding later in pregnancy usually was limited to the arcade and intercotyledonary trophoblastic cells. Binding of some lectins to cell surfaces was prominent, particularly in early pregnancy. A few lectins were excellent markers for binucleate trophoblastic cells. These distinctive surface and binucleate cell binding patterns on placentomes and endometrial epithelium are useful markers of trophoblastic cell-endometrial epithelial cell surface interactions and of binucleate cell differentiation.     

Characterization of glycoconjugates of guinea pig seminal vesicle by lectin histochemistry

In the present study, the expression of glycoconjugates in the guinea pig seminal vesicle was localized and partially characterized by lectin histochemistry using a battery of 30 different lectins specific for different carbohydrate residues. The results indicate that the glandular epithelium of the guinea pig seminal vesicle exhibits complex glycoconjugates rich in Man, -GlcNAc, -Gal, /-GalNAc, Fuc and complex NeuAc(2,6)Gal/GalNAc residues, as shown by its positive reactions to most lectins used. The Golgi region of the luminal secretory epithelial cells expresses a complex glycoconjugate pattern, as shown by its strong reactions to Man-(PSA, GNA), -GlcNAc-(S-WGA, PWA, DSA, UDA), -Gal- (RCA-I and -II), /-GalNAc-(SBA, Jac, VVA, BPA) and complex NeuAc-(SNA) specific lectins, indicating that the secretory epithelial cells are active in glycosylation and secretion process. It was also shown in the present study that the basal and luminal epithelial cells are different in their glycoconjugates. The basal epithelial cells are rich in NeuAc(2,3)Gal residues as they are stained specifically by MAA. The fibroblasts in the epithelial-smooth muscle interface and the smooth muscle cells close to the glandular epithelium are shown to express more glycoconjugates as they are stained intensely by GS-I-B4, GS-II and SBA. However, their role in the epithelial-stromal interaction in the seminal vesicle remains to be elucidated. In summary, the present study reports for the first time on the lectin binding patterns of the guinea pig seminal vesicle, and the results show that the seminal vesicle epithelium elaborates and secretes glycoconjugates in a complex pattern. Some of the lectins might be useful as histochemical markers for the secretory activity and specific structural components in the guinea pig seminal vesicle. © 1998 Chapman & Hall     

Identification of fucosylated glycoconjugates in Xenopus laevis testis by lectin histochemistry

Glycoconjugates play roles in many physiological and pathological processes. Previous works have shown important functions mediated by glycans in spermatogenesis, and the carbohydrate composition of testis has been studied by several approaches, including lectin-histochemical methods. However, the testis of Xenopus laevis, an animal model extensively employed in biochemical, cell and developmental research, has not yet been analysed. The aim of this work was to carry out a histochemical study of the fucose (Fuc)-containing glycoconjugates of Xenopus testis by means of lectins, combined with deglycosylation pretreatments. Four Fuc-binding lectins were used: orange peel (Aleuria aurantia) lectin (AAL), gorse seed (Ulex europaeus) agglutinin-I (UEA-I), fresh water eel (Anguilla anguilla) agglutinin (AAA), and asparagus pea (Lotus tetragonolobus) agglutinin (LTA), each recognizing different forms of fucosylated glycans. Labelling with UEA-I, which preferably binds Fucα(1,2) containing oligosaccharides, did not show any appreciable staining. LTA, specific for Fucα(1,3), and AAA, which binds Fucα(1,2), labelled spermatocytes and spermatids, but no labelling was seen when the histochemical procedure was carried out after either β-elimination (which removes O-linked oligosaccharides) or incubation with PNGase F (which removes N-linked oligosaccharides), suggesting that fucosylated glycans are of both N- and O-linked types. AAL, which has its highest affinity to Fucα(1,6), but also recognizes Fucα(1,2) and Fucα(1,3), labelled the whole testis, and the staining remained when the histochemical method was performed after either β-elimination or incubation with PNGase F. Labelling with AAL could be explained by the fact that this lectin could be binding to diverse fucosylated glycans in N- and O-glycans, and even in glycolipids. The importance of these glycans is discussed.     

Resuscitation of canine and feline neonates

Fetal depression following dystocia and Cesarean section has two primary causes; the first (and often most important) cause is hypoxia, and the second is depression from anesthetic agents given to the dam. Resuscitation efforts should be provided in the following order: warmth, airway, breathing, circulation, and drugs. Adequate time should be allowed for correction of hypoxia using ventilatory and circulatory support before drugs are used, with the exception of drugs given to reverse anesthetic and analgesic agents that were given to the dam prior to delivery of the neonates.     

Lectin and proteoglycan histochemistry of feline pacinian corpuscles.

We studied carbohydrate residues of glycoproteins and proteoglycans (PGs) in peritoneal Pacinian corpuscles of five adult cats. Terminal monosaccharides of glycoproteins and related polysaccharides were identified by lectin histochemistry and the PGs and glycosaminoglycans (GAGs) by specific antibodies. The most intensive lectin staining reactions indicated an abundance of glycoconjugates with terminal mannose (Man) or sialic acid residues, but no complex-type oligosaccharides were detected within the corpuscles. Terminal fucose (Fuc) and galactose (Gal) residues typical for O-linked mucin-type glycoproteins generally associated with high water binding capacity were also absent. Antibodies against unsulfated chondroitin (C-0-S), chondroitin-4-sulfate (C-4-S), and decorin showed positive reactions in the interfibrillar spaces between the lamellae, around collagen fibers, and around the lamellae of the perineural capsule, especially in the outer parts known to contain Type II collagen. Biglycan showed a preference for the innermost part of the perineural capsule (intermediate layer), known to contain Type V collagen. Collagen V and biglycan are both linked to growth processes. Hyaluronic acid (HA), chondroitin-6-sulfate (C-6-S) chains, and a chondroitin sulfate proteoglycan (CSPG) were co-localized in the terminal glia. The study of carbohydrates with high water binding capacity may contribute to our understanding of the high viscoelasticity of Pacinian corpuscles.     

Ageing of the human oviduct: lectin histochemistry

The aim of the present research was to investigate the changes of the sugar residues in the oviduct in the course of ageing in postmenopausal women vs normally menstruating women, by means of lectin histochemistry. Twenty asymptomatic postmenopausal women (48-83 years old) were recruited among patients who underwent a vaginal hysterectomy. Eight normally menstruating women were recruited as controls. Fragments of Fallopian tubes (pars ampullaris) were fixed in 10% formalin and routinely processed. The sections were labelled with HRP-lectins (PNA, SBA, DBA, WGA, Con A, LTA, UEAI). Some sections were pre-treated with neuraminidase prior to staining with HRP-lectins. Among the postmenopausal patients, our histochemical data showed that there was no difference in the localization and distribution of sugar residues of glycoconjugates as detected by various HRP-lectins. Moreover, our results demonstrated that the oviductal epithelium is characterized by apical reactivity in both ciliated and non-ciliated cells. In the course of ageing, the ciliated cells changed their morphology from bathyprismatic to large and rounded shape. ConA lectin reacted intensely with such highly degenerating ciliated cells and could be considered a marker of these cells. The degenerating ciliated cells are also characterized by the absence of sialic acid. In comparison with the sugar residues present in the control group, the oviductal epithelium of postmenopausal women is characterized by the loss of reactivity with DBA, WGA and ConA. Moreover, PNA reactive material was present at the free border of the ciliated and non-ciliated cells. The latter findings were statistically confirmed and could be considered strictly related to the ageing process.     

Expression pattern of glycoconjugates in rat retina as analysed by lectin histochemistry

The present study sought to characterize the expression and distribution of complex glycoconjugates in the rat retina by lectin histochemistry, using a panel of 21 different lectins with different carbohydrate specificities. Paraffin sections of Carnoy-fixed Sprague-Dawley rat eyes were stained with various biotinylated lectins, followed by the streptavidin-peroxidase and glucose oxidase-diaminobenzidine-nickel staining procedures. The results showed that the retinal pigment epithelium was stained intensely with LCA, Jacalin, WFA, S-WGA, PWA, DSA, UEA-I, LTA and PHA-E, suggesting that this epithelium contained glycoconjugates with alpha-Man, alpha-Glc, alpha-Gal/GalNAc, beta-GalNAc, alpha-Fuc, NeuAc and other oligosaccharide residues. The outer and inner segments of the photoreceptor layer showed different lectin binding affinities. The outer segments reacted with S-WGA and GS-II, whereas the inner segments reacted with UEA-II, UEA-I, LTA and MAA, suggesting that the inner segments contained glycoconjugates rich in alpha-Fuc and NeuAc(alpha2,3)Gal residues. PNA labelled specifically the cones and could be used as a specific marker for these photoreceptors. RCA-I, WFA, S-WGA, DSA, MAA and PHA-E reacted with both the outer and inner plexiform layers. On the other hand, UEA-I and LTA specifically labelled the outer plexiform layer, while PNA labelled the inner plexiform layer. The retinal microglial cells were labelled specifically by GS-I-B4 and SNA. Interestingly, we also observed that WFA bound specifically to Müller cells and could be used as a novel marker for this retinal glial cell. The capillaries and larger vessels in the retina and choriocapillaris reacted intensely with GS-I-B4, RCA-I, S-WGA, PWA, DSA and PHA-E. No significant differences in lectin binding were observed in the microvessels at these two sites. In summary, the present study demonstrated the expression patterns of glycoconjugates in the rat retina and that certain lectins could be used as histochemical markers for specific structural and cellular components of the rat retina.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Inherited abnormalities of platelet glycoproteins

Glanzmann's thrombasthenia and the Bernard-Soulier syndrome are inherited blood disorders characterized by abnormalities in different aspects of platelet function during haemostasis. Platelets from patients with thrombasthenia do not aggregate in response to the normal physiological platelet aggregation inducing stimuli, while Bernard-Soulier platelet have a reduced capacity to adhere to exposed subendothelium. Deficiencies of different membrane glycoproteins have been located in the platelets of both disorders and suggest specific roles for membrane glycoproteins in different aspects of platelet function.     

Medical management of canine and feline dystocia

When dystocia is diagnosed in the bitch or queen, two forms of treatment exist: medical or surgical therapy. Medical management of dystocia has the advantage of aiding completion of the parturition process without surgery or anesthesia. However, since not all cases of dystocia can be managed medically, educated and careful decision making is required prior to instituting medical management in cases of dystocia. Improper medical treatment, especially when surgical management is clinically indicated, can result in compromise and even death of the dam and fetuses. This paper focuses on the decision making necessary prior to instituting medical management for cases of dystocia in both bitches and queens, and describes available therapeutics.     

Surgical management of canine and feline dystocia

If medical management of dystocia has failed or is inadvisable, a Cesarean section is indicated. The necessity of surgery is primarily based on the condition of the dam, progression of labor, and fetal heart rate. Timely intervention is crucial for optimal fetal and maternal survival. Surgical technique may vary, based on the needs of each individual case. There are many options for each portion of the surgery, including the choice of anesthetic protocol, abdominal approach, uterine incision location, and post-surgical pain management. Indications for surgery and some of the options for each step of the procedure are presented. Episiotomy is rarely used to treat dystocia and therefore, it is discussed only briefly.