Sequential Changes in Aberrant Crypt Foci and Lectin Expression in the Early and Late Stages of DMH-Induced Colon Carcinogenesis in Rats

BACKGROUND/AIMS: The purpose of this study was to investigate the malignant potential of aberrant crypt foci (ACF) by measuring the multiplicity of crypts and lectin expression in the early and late stages of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. METHODS: Six-week-old Wistar rats were injected subcutaneously with DMH for 27 weeks. We classified ACF according to the number of crypts per ACF as a few crypts ( or =4 crypts, NC ACF). Immunohistochemistry was used to evaluate lectin expression. RESULTS: In the early stage, FC ACF (590/1,902, 31.0%) occurred more frequently than NC ACF (35/449, 7.8%); whereas in the late stage, NC ACF (176/449, 39.2%) occurred more frequently than FC ACF (324/1,902, 17.0%). The number of ACF peaked at 15 to 20 weeks. The ratio of NC/FC ACF increased gradually during carcinogenesis. The expression of both UEA1 and PNA was higher in NC ACF than FC ACF. Lectin expression increased in the late stage compared with the early stage. CONCLUSIONS: The expression of lectin was higher in NC ACF and ACF in the late stage. Therefore, ACF with higher multiplicities in the late stage may have more malignant potential in DMH-induced colon carcinogenesis.

Evaluation of acrosomal status and sperm viability in fresh and cryopreserved specimens by the use of fluorescent peanut agglutinin lectin in conjunction with hypo-osmotic swelling test

OBJECTIVE: In this study, we evaluated whether the hypo-osmotic swelling test (HOST) can be used as a vital marker in combination with peanut agglutinin (PNA) - labeling in fresh and cryopreserved spermatozoa. MATERIALS AND METHODS: Human sperm populations were exposed to a hypo-osmotic medium for 60 minutes, and then incubated in a 1 µg/mL solution of the fluorescent dye Hoescht 33258 (H33258) for 10 minutes. Excess stain was removed by washing in phosphate-buffered saline (PBS) solution, and the pellet was resuspended in 100 µL of culture medium. Twenty microliters of this solution were subsequently smeared on a microscope slide, and fixed in ice-cold methanol to permeabilize the sperm membranes. The fixed smears were finally incubated in a 40-µg/mL FITC-PNA solution for 20 minutes. Simultaneous assessment of acrosome and viability scores was done in a fluorescent microscope equipped with appropriate filters and phase contrast illumination. The same slide was examined for FITC-PNA labeling, tail swelling, and for Hoechst-33258 staining by interchanging the filters and phase contrast optics. RESULTS: In fresh specimens, HOST was found to provide viability assessments comparable to those obtained using the H33258 method (r = 0.95). However, the results of HOST and H33258 were not correlated in cryopreserved specimens (r = 0.22). There was no alteration of PNA-labeling due to the HOST or H33258. CONCLUSIONS: FITC-PNA labeling in conjunction with the visualization of the morphological change induced by exposure to hypo-osmotic solution provides a simple but effective method for establishing the state of acrosomal membrane and viability in fresh human spermatozoa, but this technique is not reliable for cryopreserved ones.

Dynamic light scattering study of peanut agglutinin: size, shape and urea denaturation.

Peanut agglutinin (PNA)is a homotetrameric protein with a unique open quaternary structure. PNA shows non-two state profile in chaotrope induced denaturation. It passes through a monomeric molten globule like state before complete denaturation (Reddy et al 1999). This denaturation profile is associated with the change in hydrodynamic radius of the native protein. Though the molten globule-like state is monomeric in nature it expands in size due to partial denaturation. The size and shape of the native PNA as well as the change in hydrodynamic radius of the protein during denaturation has been studied by dynamic light scattering (DLS). The generation of two species is evident from the profile of hydrodynamic radii. This study also reveals the extent of compactness of the intermediate state.

Effect of caspase inhibitors on the post-thaw motility, and integrity of acrosome and plasma membrane of cryopreserved equine spermatozoa.

The present study was designed to test the hypothesis that addition of anticaspase cocktails (inhibiting caspases and thus blocking apoptosis) to the extenders increases the post-thaw viability of equine spermatozoa. The addition of caspase inhibitors failed to improve the acrosome and plasma membrane integrity of spermatozoa, suggesting that in equine sperm cryopreservation protocols, the addition of these caspase inhibitors to cryopreservation medium may not be beneficial in protecting the sperm from the stress of cryopreservation.

Preparation of 5-fluorouracil-peanut agglutinin conjugate and determination of the ratio of drug to conjugate / 生物医学工程学杂志

5-fluorouracil was combined with peanut agglutinin by a water-soluble carbodiimide to prepare the tumor target conjugate of 5-fluorouracil-peanut agglutinin and the ratio of drug to conjugate was determined by the modified trinitrobenzenesulfonic acid method (TNBS). The ratio of drug to conjugate was 76.33%. The result showed that 5-fluorouracil could link to the peanut agglutinin by EDC/NHS crosslinking with high drug ratio.

Histochemistry of Six Lectins in the Tissues of the Flat Fish Paralichthys olivaceus

Lectins are glycoproteins that specifically bind carbohydrate structures and may participate in the biodefense mechanisms of fish. In this study, the binding of three lectins, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Bandeiraea simplicifolia BS-1 (isolectin B4), Triticum vulgaris (WGA), Arachis hypogaea (PNA) and Ulex europaeus (UEA-I) were studied in the gill, liver, intestine, kidney, heart, and spleen of the flat fish Paralichthys olivaceus. DBA was detected in intestinal mucous cells, as well as in gill epithelial and mucous cells. It was weakly detected in renal tubule epithelial cells and in bile duct epithelial cells. The strong SBA staining was seen in the intestinal club cells, in bile duct epithelial cells and renal tubule epithelial cells. There were intense positive reactions for isolectin B4 in gill epithelial and mucous cells, and the strong isolectin B4 staining was seen in epithelial cells of the bile duct and intestine. The strong WGA staining was seen in the gill mucosal cells, sinusoid, renal tubule epithelial cells and mucosal cells of the intestine. UEA-I was detected in the gill epithelial and mucosal cells, bile duct epithelial cells and renal tubular epithelial cells. These results suggest that the six lectins examined were localized in the covering epithelia of the various organs of the flat fish and they may participate in the biodefense mechanism of the intra body surface in which is exposed to various antigens.

Electron Microscopic Demonstration of Sialoglycoconjugates in the Sinus Mucosa of Rabbits after Inoculation of the Influenza A Virus

This study was conducted in order to observe ultrastructural changes in the expression of sialoglycoconjugates in maxillary sinus mucosa after inoculation of influenza A virus utilizing four different gold-labeled lectins : ckia amurensis (MAA), wheat germ agglutinin (WGA), sambucus nigra (SNA), and peanut agglutinin (PNA). A comparison of the affinities of these gold-labeled lectins demonstrated the varying distributions of sialoglycoconjugates in the ciliary layer and the granules in goblet cells. Examination of normal sinus mucosa labeled with four gold-labeled lectins showed the distribution of sialoglycoconjugates to be mainly in the ciliary layer and the granules in goblet cells and restricted to the surface of the cilia, microvilli and the secretory light granules. The application of an influenza A virus infection decreased the labeling intensity of gold-labeled MAA in the cilia and the secretory granules but not of WGA. SNA gold did not label the surface of the cilia and granules in either case. PNA gold particles, however, labeled the cilia and the secretory granules very weakly in normal sinus mucosa, but labeled moderately in cases of influenza A virus infection. These results suggest that the sugar residues of sialoglycoconjugates consist of Neu5Ac(alpha2, 3)Gal, GlcNAc, Neu5Ac. They also suggest that the sugar residues serve as a protecting factor or modulator against influenza A virus infection.

Vegetative tissue lectins of peanut (A. hypogaea).

Lectin activities in roots, nodules, stems and leaves of 1-6 week old peanut plant (A. hypogaea) were checked by erythrocyte (human and rabbit) agglutination and sugar inhibition assays. Human and rabbit erythrocyte agglutinating activities were specifically inhibited by lactose/cellobiose (SLII) and methyl alpha-mannoside (SLI) respectively. Seeds, embryos and cotyledons agglutinated neuraminidase treated human erythrocytes and that activity was inhibited by T-disaccharide. In the roots of field grown plants SLI was the major activity, while nodules showed both activities (SLI and SLII). Specific activities of SLI and SLII were maximal in stem tissue and hypocotyl exhibited minimal levels. Actively growing tissues like newly emerging young leaves and elongating stem contained more SLII activity in comparison to the mature tissues. Immunological test indicated that all the vegetative tissue lectins are serologically related.

A Case of Polyagglutination due to T Activation / 대한수혈학회지

Red blood cells that agglutinate with most normal adult sera but never with own sera are termed polyagglutinable and can be separated by patterns of lectin reactivity into various types. Among these polyagglutination, activation of the T cryptantigen occurs when carbohydrate structures on glycophorins A and B lose sialic acid and express the disaccharide Gal beta-l-3 GalNac which reacts with the peanut agglutinin, a lectin from Arachis hypogaea. T activation is a temporary condition due to exposure of the membrane antigen to the action of microbial neuraminidase. In T activated red cells, the following hazards, which are theoretically possible, are spontaneous polyagglutination of red cells in vitro, in vivo and severe blood transfusion reactions. We experienced a case of T activation in 6 month old girl with bacterial meningitis caused by Streptococcus pneumoniae. The reactivity to lectins indicated the patient's red cells were T activated. We report a case of T activation in an infant with the review of literature.

Peanut agglutinin binding by gastric mucosal epithelial cells in Helicobacter pylori associated gastritis.

Gastric biopsies (42) from patients with peptic ulcer disease were classified into Helicobacter pylori positive (32) and negative (10) groups, based on the results of tissue urease test and microscopic demonstration of spiral bacteria. A statistically significant difference in peanut agglutinin (PNA) binding between the two groups was observed, attributable to exposure of sialic acid residues on gastric epithelium in the H. pylori positive group. That the negative binding was due to sialic acid, was further confirmed by application of sialidase digestion technique. These results support the existing biochemical evidence for exposure of sialic acid residues on H. pylori colonized epithelium.

Significance of Peanut Agglutinin in the Differentiation between Nevocellular Nevus and Malignant Melanoma / 대한피부과학회지

Using peanut agglutinin(PNA), neurarninidase, and avidin-biotin peroxidase com plex(ABC) technique, normal skin specimens, nevocellular nevi, and malignant melanomas were studied, and different PNA binding patterns between nevocellular nevi and malignant melanomas were observed. The results were as follows : 1. In normal skin, except the basement membrane, epidermis and hair follicle epithelium showed a cell membrane staining of PNA after neuraminidase pretreat ment. Sebaceous glands revealed membranous and cytoplasmic staining of PNA, but sweat ducts were not stained. 2. In nevocellular nevi, none of the nevus cells were stained with PNA aftet neuraminidase preteatment. 3. In malignant melanomas, all of the melanorna cells were stained along the cell mernbrane with PNA after neuraminidase pretreatment. Therefore, the PNA staining after neuraminidase pretreatment on paraffin embedded sections using ABC technique is considered to be a useful probe for the differentiation between malignant malanoma and nevocellular nevus.

Lectin binding in colorectal mucosa.

Lectin binding of goblet cell mucin in human colonic mucosa was studied in patients with irritable bowel syndrome, colorectal malignancy and ulcerative colitis using plant lectins, Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA). Normal colonic mucosa demonstrated a strong binding with DBA (100%) but did not bind to PNA at all. Colonic carcinomas showed strong PNA binding (7 of 15 biopsies) while DBA binding was absent in 14 of 15 biopsies. The transitional mucosa showed reduced or absent DBA binding in 6 and positive PNA binding in 2 of 15 biopsies. During the active phase of ulcerative colitis, there was a loss of DBA binding in 10 of 15 biopsies, which was restored during remission in all. One biopsy with severe dysplasia showed PNA binding. It is concluded that normal colorectal mucosa binds DBA strongly but not PNA. Malignant tissue and transitional mucosa reveal PNA binding often, with decreased DBA binding. In ulcerative colitis DBA binding decreases during phases of activity.

Significance of Peanut Agglutinin in the Differentiation between Basal Cell Carcinoma and Trichoepithelioma / 대한피부과학회지

Great difficulty may be encountered in the differentiation of basal cell carcinoma from trichoepithelioma snd, in some cases, it may even be irnpossible. Immunohistochemical methods using peanut agglutinin(PNA) which is glycoprotein of non-immune origin selectively binding to galactose-N-acetyl-galactosa-mine are increasingly used in dermatopathology to improve the diagnosis and differential diagnosis. Using PNA, anti-PNA antibody, and peroxidase antiperoxi-dase(PAP) technique, normal skin specimens, basal cell carcinomas, trichoepitheliiomas, and a variety of different skin tumors were studied, and different PNA Ibinding sites between basal cell carcinomas and trichoepitheliomas were observed. The results were as follows : l. In normal skin, except the basement membrane, epidermis and hair follicle epithelium showed a cell membrane staining of PNA, which stained weakly in the Ibssal cell layer. Sebaceous glands revealed membranous and cytoplasmic staining of PNA, but sweat ducts and duct coi1s were mostly negative. 2. 34 of 36(94.4%) basal cell carcinoma sections demonstrated peritumorous PNA-positive bands, and none of 5 trichoepithelioma sections showed peritumorous PNA-binding. 3. Peritumorous PNA-positive bands were strongly positive in solid and keratotic basal cell carcinomas, but decreased or absent in the vicinity of the ulceration or the dense inflammatory infiltration. 4. None of the other skin tumors(squamous cell carcinoms, keratoscanthoma, Bowens disesse and actinic keratosis} showed a periturnorous PNA-positive band. Therefore, we believe that the PNA staining on paraffin-embedded sections using PAP technique can be a useful probe for the differentiation of basal cell carcinoma from trichoepithelioma.