Glycoprotein Staining Research Articles

Protein-Bound Polysaccharide K (PSK)-Rich Extract of Lentinus Polychrous Exhibits Anticancer Activity by Apoptosis Induction and Cell Cycle Arrest in Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma of the bile duct epithelial. There is no standard regimen yet for patients with adequate performance status. Therefore, effective agents are necessary. Recently, protein-bound polysaccharide K (PSK) has shown a great anticancer potential. L. polychrous is a Thai local edible mushroom that traditionally used as a folk medicine for treatments of fever and inflammation. This study aimed to identify the PSK from hot water extract of L. polychrous and examine the anticancer activity against a CCA cell line KKU-213A. The PSK were quantified by ELISA and characterized using SDS-PAGE, glycoprotein staining kit, and western blot analysis. The antioxidant activity was assessed by DPPH assay. Cytotoxic effect against KKU-213A was assessed by MTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. The highest PSK levels were found in L. polychrous extract compared with the other 14 mushroom species which purified PSK is approximately 70 kDa of glycoprotein. The hot water extract of L. polychrous showed strong antioxidant activity. The cell viability of KKU-213A cells was significantly inhibited with L. polychrous extract at an IC50 value of 3.31 ± 0.09 mg/mL. In addition, the induction of cell cycle arrest at S and G2/M phases was observed together with induction of apoptosis, and mitochondrial membrane dysfunction in concentration-dependent manner. Hence the L. polychrous extract is the great source of PSK with strong antioxidant activity and exhibits anti-CCA effects through cell cycle arrest and apoptotic induction. HIGHLIGHTS Lentinus polychrous, a Thai local edible mushroom, is the good source of PSK and powerful antioxidant activity. The hot water extract of L. polychrous suppresses cell viability, induces apoptosis, cell cycle arrest, and mitochondrial dysfunction in CCA cell line. The anticancer effects of L. polychrous extract against CCA cells makes it a potential agent for CCA treatment. GRAPHICAL ABSTRACT

Histochemical and ultrastructural evaluation of myopic corneal lenticules based on refractive error.

This study aimed to investigate cell degeneration, apoptosis, and ultrastructural differences in refractive lenticules (RL) obtained using small incision lenticule extraction (SMILE) compared with spherical equivalence (SE) refraction values. This study included 84 eyes from 42 patients. Patients were divided into two groups according to the SE values: those with values below 4 diopters (D) (Group 1) and above 4 diopters (D) (Group 2). Patients who did not belong to the same SE group were excluded from the study. One RL obtained from each patient was separated for light microscopy and immunohistochemical examinations, and another for transmission electron microscopy (TEM) examinations. Caspase-3 for apoptosis and alpha-smooth muscle actin (α-SMA) for cell degeneration were evaluated using immunohistochemical examinations. Histological analyses showed that the density of collagen fibres was greater in Group 1 than in Group 2. Glycoaminoglycan and glycoprotein staining intensities were also higher in Group 1. TEM observations showed that Group 1 had intact cell and nuclear membranes, peripheral heterochromatin, and large nuclei, while Group 2 showed heterochromatin condensation and fragmentation, increased intracellular vacuoles, and loss of cytoplasm. Immunohistochemical analyses revealed that α-SMA and caspase-3 were significantly higher in Group 2 than in Group 1 (p < 0.001 and p < 0.001, respectively). Cell degeneration and apoptosis were significantly more common in the RLs with high SE values after SMILE surgery. The tissue response induced by surgery was more severe in the RLs with high SE values. This should be considered when reusing RLs.

Glycoprotein from Sargassum fusiforme exhibiting anti-inflammatory responses in vitro and in vivo via modulation of TLR4/MyD88 and NF-κB signaling

This study focuses on the identification and characterization of a glycoprotein from Sargassum fusiforme (Harvey) Setchell (SFGP), as well as investigating its potential anti-inflammatory properties both in vitro and in vivo, along with the underlying mechanism. SDS-PAGE analysis revealed a prominent band with a molecular weight of <10 kDa, consisting of 58.39 % protein and 41.61 % carbohydrates, which was confirmed through glycoprotein staining and Coomassie blue staining. Various analytical techniques, including high-resolution mass spectrometry (HRMS), FTIR, amino acid analysis, and UV–visible spectrometry, provided evidence for the presence of monosaccharides (such as d-glucose and mannose) and 17 amino acids linked by an O-glycopeptide bond.In vitro and in vivo studies were conducted to assess the anti-inflammatory activities of SFGP. The results demonstrated that SFGP effectively attenuated nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in LPS-treated RAW264.7 cells. Moreover, SFGP administration significantly and dose-dependently suppressed TLR4/MyD88 signaling as well as the phosphorylation of MAPKs, IκB, and NF-κB, leading to a reduction in the production of TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW264.7 cells. Furthermore, the anti-inflammatory efficacy of SFGP was validated in a carrageenan-induced inflammatory mouse model. These findings indicate that SFGP exhibits anti-inflammatory characteristics and has the potential to be utilized as a novel anti-inflammatory agent.

Optimization and application of silver staining of non-glycosylated and glycosylated proteins and nucleic acids for agarose native gel electrophoresis

Electrophoresis is one of the major techniques to analyze macromolecular structure and interaction. Its capability depends on the sensitivity and specificity of the staining methods. We have here examined silver staining of proteins and nucleic acids separated by agarose native gel electrophoresis. By comparing five commercial kits, we identified Silver Stain Plus from Bio-Rad most adequate, as it provided little background staining and reasonable band staining. One of the disadvantages of the Silver Stain Plus kit is its variable staining of glycoproteins as tested with several model samples, including hen egg white proteins, α1-acid glycoprotein and SARS-CoV-2 Spike protein.One of the advantages of silver staining is its ability to stain nucleic acids as demonstrated here for a model nucleic acid with two kits. It was then used to monitor the removal of nucleic acids from the affinity-purified maltose binding protein and monoclonal antibody. It also worked well on staining proteins on agarose gels prepared in the vertical mode, although preparation of the vertical agarose gels required technological modifications described in this report. With the silver staining method optimized here, it should be possible in the future to analyze biological samples that may be available in limited quantity.

Infection of bursal disease virus abrogates the extracellular glycoprotein in the follicular medulla

In the medulla of bursal follicle, only the secretory dendritic cell (BSDC) is furnished with secretory machinery. The granular discharge of BSDC appears in membrane-bound and solubilized forms. Movat pentachrome staining proves that the solubilized form is a glycoprotein, which fills up the extracellular space of follicular medulla. The glycoprotein contributes to bursal microenvironment and may be attached to the surface of medullary lymphocytes. The secretory granules of BSDC may be fused, resulting in large, irregular dense bodies, which are the first sign of BSDC transformation to macrophage-like cells (Mal). To determine the effect of infectious bursal disease virus (IBDV) infection on the extracellular glycoprotein and BSDC, SPF chickens were experimentally infected with IBDV. On the surface of BSDC, the secretory substance is in high concentration, which may contribute to primary binding of IBDV to BSDC. The early distribution of IBDV infected cells is in consent with that BSDC. The IBDV infected BSDC rapidly transforms to Mal in which the glycoprotein staining appears. In the dense bodies, the packed virus particles inhibit the virus particles preventing the granular discharge, which may represent the first, early phase of virus replication cycle. The absence of extracellular glycoprotein results in alteration in the medullary microenvironment and subsequently B cell apoptosis. On the surface of medullary B cells, the solubilized secretory substance can be in much lower concentration, which results in secondary binding of IBDV to B cells. In secondary, late phase of virus replication cycle, the virus particles are not packed in electron dense substance which results in cytolytic lymphocytes and presence of virus in extracellular space. The Mal emigrates into the cortex, where induces inflammation, recruiting heterophil granulocyte and monocyte.

A membrane protein insertion complex

The present study examined the long-standing concept that changes in hippocampal circuitry contribute to age-related learning impairment. Individual differences in spatial learning were documented in young and aged Long–Evans rats by using a hippocampal-dependent version of the Morris water maze. Postmortem analysis used a confocal laser-scanning microscopy method to quantify changes in immunofluorescence staining for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the principal relays of hippocampal circuitry. Comparisons based on chronological age alone failed to reveal a reliable difference in the intensity of SYN staining in any region that was examined. In contrast, aged subjects with spatial learning deficits displayed significant reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM) relative to either young controls or age-matched rats with preserved learning. SYN intensity values for the latter groups were indistinguishable. In addition, individual differences in spatial learning capacity among the aged rats correlated with levels of SYN staining selectively in three regions: outer and middle portions of the dentate gyrus molecular layer and CA3-LM. The cross-sectional area of SYN labeling, by comparison, was not reliably affected in relation cognitive status. These findings are the first to demonstrate that a circuit-specific pattern of variability in the connectional organization of the hippocampus is coupled to individual differences in the cognitive outcome of normal aging. The regional specificity of these effects suggests that a decline in the fidelity of input to the hippocampus from the entorhinal cortex may play a critical role.

Light microscopic study on the absorptive cells and goblet cells in the intestine of adult common carp Cyprinus carpio

The purpose of this study was to describe of some histological structures and histochemical features of the absorptive cells and goblet cells in intestine of common carp. In this study, Fifteen adult male common carp were catching alive from the AL-Forat river, with age about (7 - 12) months and mean of their weight was (2100 ± 81 g) and mean standard length was (50.4 ± 3.1 cm), immediately after death.Incision was made through the midventral line of the fish just from cranial to the anus to expose the intestine, specimens of intestine were taken and washed with (0.9 %) normal saline solution. Ten samples were obtained from different regions of each portion of the intestine (anterior, middle and posterior), fixed by either 10% neutral buffered formalin or Bouin’s solution approximately 24 hours at room temperature and then treated by routine histological processing. The stains were used, Hematoxylin and Eosin, periodic acid Schiff, Alcian blue pH 2.5, and Combinated Alcian blue PH 2.5 plus periodic acid Schiff. The Mean number, height and width of mucosal folds were measured and counts of goblet cells in the mucosal folds of each portion of the intestine. The results showed that the intestinal epithelium is simple columnar, the enterocytes were tall columnar cells with brush border; goblet cells were ovoid, located between the enterocytes. The folds composed of the entire thickness of the mucosa, mean number, height and width of the mucosal folds were few in posterior portion of intestine compare with that in other portions. The anterior portion of the intestine has largest number of mucosal folds, while has less number of goblet cells compare to middle and posterior portions of the intestine. The epithelial cells stained red-purple by periodic acid Schiff stain with Alcian blue, the goblet cells were take positive reaction with periodic acid Schiff stain, and with Alcian blue plus periodic acid Schiff, while negative with only Alcian blue stain. Glycoprotein staining intensity varied from very weak to moderate, whereby the presence of neutral, acidic and sulphatedglycoconjugates. In conclusion, numbers of goblet cells were observed to be increased towards posterior portion of intestine required as lubricant for fecal expulsion, and reversed relation between number of the folds and goblet cells.

Fact or Fiction, It Is Time for a Verdict on Vasculogenic Mimicry?

The term vasculogenic mimicry (VM) refers to the capacity of certain cancer cells to form fluid-conducting structures within a tumor in an endothelial cell (EC)-free manner. Ever since its first report by Maniotis in 1999, the existence of VM has been an extremely contentious issue. The overwhelming consensus of the literature suggests that VM is frequently observed in highly aggressive tumors and correlates to lower patient survival. While the presence of VM in vivo in animal and patient tumors are claimed upon the strong positive staining for glycoproteins (Periodic Acid Schiff, PAS), it is by no means universally accepted. More controversial still is the existence of an in vitro model of VM that principally divides the scientific community. Original reports demonstrated that channels or tubes occur in cancer cell monolayers in vitro when cultured in matrigel and that these structures may support fluid movement. However, several years later many papers emerged stating that connections formed between cancer cells grown on matrigel represented VM. We speculate that this became accepted by the cancer research community and now the vast majority of the scientific literature reports both presence and mechanisms of VM based on intercellular connections, not the presence of fluid conducting tubes. In this opinion paper, we call upon evidence from an exhaustive review of the literature and original data to argue that the majority of in vitro studies presented as VM do not correspond to this phenomenon. Furthermore, we raise doubts on the validity of concluding the presence of VM in patient samples and animal models based solely on the presence of PAS+ staining. We outline the requirement for new biomarkers of VM and present criteria by which VM should be defined in vitro and in vivo.

Cover Caption

June Online Cover: Glycoprotein staining of six chickpea types; chickpea seeds and their final products, from “Quality of a Chickpea‐Based High Protein Snack” by Sahil Gupta, Changqi Liu, and Shridhar K. Sathe. p. 1621.

The impact of the molecular weight of dextran on formation of whey protein isolate (WPI)-dextran conjugates in fibers produced by needleless electrospinning after annealing.

The conjugation reaction of electrospun fibers of a mixture of whey protein isolate (WPI) and dextran using different molecular weights (40, 70, and 100 kDa) and mixing ratios was studied. This study includes the electrospinnability of a mixture of WPI and dextran, and the conjugation reaction between them via the initial stage of the Maillard reaction. The WPI-dextran fibers were characterized using optical and transmission electron microscopy. The covalent attachment of dextran to WPI was confirmed using sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis with protein and glycoprotein staining. Both 70 and 100 kDa of dextran and WPI at mixing ratios of 2 : 1 and 3 : 1 in phosphate buffer (30 mM, pH 6.5) were electrospun using needleless electrospinning. The solution concentration of the mixture was 50 wt% (33.3/37.5 wt% for dextran/16.5/12.5 wt% for WPI). The optimal conjugation conditions chosen from the experiments were a mixture of dextran (70 kDa)-WPI at 3 : 1 (75% relative humidity, 60 °C, 48 h).

Anti-pandemic influenza A (H1N1) virus potential of catechin and gallic acid

BackgroundThe pandemic influenza A (H1N1) virus has spread worldwide and infected a large proportion of the human population. Discovery of new and effective drugs for the treatment of influenza is a crucial issue for the global medical community. According to our previous study, TSL-1, a fraction of the aqueous extract from the tender leaf of Toonasinensis, has demonstrated antiviral activities against pandemic influenza A (H1N1) through the down-regulation of adhesion molecules and chemokine to prevent viral attachment. MethodsThe aim of the present study was to identify the active compounds in TSL-1 which exert anti-influenza A (H1N1) virus effects. XTT assay was used to detect the cell viability. Meanwhile, the inhibitory effect on the pandemic influenza A (H1N1) virus was analyzed by observing plaque formation, qRT-PCR, neuraminidase activity, and immunofluorescence staining of influenza A-specific glycoprotein. ResultsBoth catechin and gallic acid were found to be potent inhibitors in terms of influenza virus mRNA replication and MDCK plaque formation. Additionally, both compounds inhibited neuraminidase activities and viral glycoprotein. The 50% effective inhibition concentration (EC50) of catechin and gallic acid for the influenza A (H1N1) virus were 18.4 μg/mL and 2.6 μg/mL, respectively; whereas the 50% cytotoxic concentrations (CC50) of catechin and gallic acid were >100 μg/mL and 22.1 μg/mL, respectively. Thus, the selectivity indexes (SI) of catechin and gallic acid were >5.6 and 22.1, respectively. ConclusionThe present study demonstrates that catechin might be a safe reagent for long-term use to prevent influenza A (H1N1) virus infection; whereas gallic acid might be a sensitive reagent to inhibit influenza virus infection. We conclude that these two phyto-chemicals in TSL-1 are responsible for exerting anti-pandemic influenza A (H1N1) virus effects.

A simple, time-saving, microwave-assisted periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels.

We introduce an optimized periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid-Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave-assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave-accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.

EPCAM Expression in Colon Adenocarcinoma and its Relationship with TNM Staging.

Background:Since epithelial cell adhesion molecule glycoprotein (EPCAM) is associated with the development and metastasis of colon adenocarcinoma, it can be helpful in predicting the tumor stage before surgery. In this study, we investigated EPCAM glycoprotein expression in colon adenocarcinoma and its relationship with tumor staging.Materials and Methods:This study was done on formalin-fixed and paraffin-embedded tissues of 71 patients diagnosed with colon adenocarcinoma, together with normal tissues around them, which were available at the archive of pathology lab of Al-Zahra hospital, Isfahan. Hematoxylin and eosin (HandE) and immunohistochemistry (IHC) staining methods for EPCAM marker were performed on paraffin-embedded blocks.Results:The percentage of staining of EPCAM glycoprotein in the tumoral and normal tissues of 71 patients with colon adenocarcinoma was studied and compared. In normal tissue, 84.5% showed strong staining, 15.5% showed moderate to strong, and none of the patients showed moderate, weak, or no staining at all. In the tumoral tissue, 25.4% had strong, 25.4% had moderate to strong, 26.8% showed moderate, 16.9% showed weak and 5.6% had no staining at all. EPCAM expression was significantly less in tumoral tissue than in normal.Conclusion:There was an inverse relationship between tumor staging and the percentage of staining in EPCAM glycoprotein so this marker can be used for predicting the tumor stage.

Exploring the oviductal fluid proteome by a lectin-based affinity approach.

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein, we assessed the combined use of Concanavalin A and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound as well as 130 lectin-bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin-bound fraction were known glycoproteins (p-value [FDR] = 1.45E-31). The low variance of the number of peptide spectrum matches for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3% for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF.

Sialic acid-specific lectin participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus merguiensis.

A sialic acid-specific lectin was purified from the hemolymph of Fenneropenaeus merguiensis by repetitive affinity fetuin-agarose column chromatography. The purified F. merguiensis lectin (called FmL) consisted of two distinct 30.9 and 32kDa subunits with identical N-terminal amino acid sequences of ten residues. FmL was also composed of sugar moieties; glucosamine, glucose, mannose and N-acetyl neuraminic acid but not N-glycolyl neuraminic acid. It was postulated to be a glycoprotein as it was positively stained by glycoprotein staining kit and detected by some bionylated plant lectins. Deglycosylation by either peptide N-glycosidase F or trifluoromethanesulfonic acid turned both types of FmL subunits to 28kDa peptides. The internal peptide sequence of FmL was similar to a fibrinogen-related domain of human ficolin and the horseshoe crab lectin. Determination of the lectin concentrations in the hemolymph was performed by ELISA while its hemaglutinating activity (HA) was tested by hemagglutination. Both specific lectin concentrations and HA increased as shrimp developed ovarian maturation stages 2 to 4. Their constitutive levels were found in pre-vitellogenic females and higher than those of males. Both specific lectin concentrations and HA of FmL were inducible to the highest levels at 12h after F. merguiensis was challenged by pathogenic Vibrio harveyi. The FmL-induced agglutination of V. harveyi was specifically abolished by sialic acid, fetuin and bacterial cell wall components. These findings might indicate the implication in an immune response of FmL to protect the shrimp themselves or their spawning eggs towards pathogenic bacteria in surrounding environment.

Oxidative polyaldehyde production: a novel approach to the conjugation of dextran with soy peptides for improved emulsifying properties.

Dextran (polyol) was oxidized with 0, 0.5, 1, and 2% sodium hypochlorite at pH 9.5 and 35°C to produce polyaldehyde dextran (PD), which was subsequently conjugated with soy peptides (SP) to improve surface activity. SP-PD complexes were formed by heating 1% SP and 10% PD at 60°C and pH 6.5 for 48h. PD was more reactive than unmodified dextran with SP to produce conjugates based on the Schiff base with absorption at 294nm. The formation of SP-PD complexes was confirmed by SDS-PAGE with glycoprotein staining. Turbidity and particle size measurements indicated the SP-PD conjugates had significantly improved emulsifying properties compared to non-conjugated SP and the SP/PD mixtures. The results indicate that controlled oxidation of polysaccharides can be a novel technique to efficiently synthesize amphiphilic functional biopolymers.

Study on isolation and determination of glycoprotein from tea plant leaves

Tea is a healthy food, because it has various bioactive components. Recent research indicated that tea glycoprotein might be a bioactive component in tea. In this report, total proteins were extracted from tea fresh leaves, and coarse glycoproteins were isolated through the methods of ammonium sulfate fractions (45%--70%) and Sephadex G-100 chromatography. According to control staining of glycan and polypeptide, glycoproteins were determinated by using Pierce GelCode Glycoprotein Staining Kit and Coomassie Brilliant Blue R250, and three tea glycoproteins were fast purified from the SDS-gels. Sixteen native bioactive glycoproteins were gained from tea total proteins by ConA-Sepharose 4B affinity chromatography.

The effect of Dermatophagoides pteronyssinus group 7 allergen (Der p 7) on dendritic cells and its role in T cell polarization

The innate signaling pathway for Th2 immunity activated by inhaled allergens has not been well defined. Dendritic cells (DCs) can use their innate pattern-recognition Toll-like receptors and C-type lectin receptors to generate innate immunity and influence adaptive responses. The aim of this study was to investigate the glycoform of Dermatophagoides pteronyssinus group 7 allergen (Der p 7) and its functional interaction with dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). Both bone marrow-derived dendritic cells (BMDCs) derived from mice and monocyte-derived DCs (MDDCs) derived from human peripheral blood mononuclear cells (PBMCs) were used to investigate the function of Der p 7. Dendritic cells derived from THP1 were used to investigate the glycol form of Der p 7. Der p 7 interacted with DC-SIGN as recognized by immunoprecipitation and glycoprotein staining, and its function was partially inhibited by deglycosylation. When BMDCs were cultured with Der p 7, the secretion of IL-6 and gene expressions of IL-6, OX40L and Jagged-1 were increased. IL4+/CD4+ T cells could be induced by Der p 7, however IFN-γ+/CD4+ T cells were down-regulated. The effects of Der p 7 on DCs were down-regulated in the presence of DC-SIGN or TLR4 antibodies and deglycosylation. rDer p 7 enhanced the DC differentiation of CD80 and CD86. The effects of Der p 7 pulsed-MDDCs on IL4+/CD4+ and IFN-γ+/CD4+ T-cell differentiation of human PBMCs were significantly increased after Der p 7 stimulation and decreased by DC-SIGN antibody inhibition. In conclusion, the Der p 7 is a glycoprotein and its function was partially through glycan binding. BMDCs could be activated by Der p 7 through TLR4 and DC-SIGN, and followed by the expression of OX40 ligand (OX40L) and Jagged-1, the expressions of IL4+/CD4+ cells were enhanced. These findings identify a previously unrecognized function of Der p 7 and establish a link between innate TLR4/C-type lectin receptors and adaptive Th2 immunity.

Extracellular matrix in oral squamous cell carcinoma: Friend or foe?

Oral squamous cell carcinoma (OSCC) primarily spreads through direct invasion and/or lymphatic route. During the invasion, tumor cells break through the basement membrane, penetrate the connective tissue to interact with the extracellular matrix (ECM). An attempt was made to evaluate the connective tissue changes in different grades of OSCCs and their influence in predicting the biological behavior of these tumors. A total of 30 histologically proven cases comprising 5 normal mucosa, 10 well-differentiated OSCC's, 10 moderately differentiated OSCC's, and 5 poorly differentiated OSCC's were examined for the presence of any ECM changes by using special stains. Interpretation of staining intensity was carried out and statistically analyzed. Van Gieson stain showed abundant thick collagen fibers, dispersed collagen fibers, thin few dispersed collagen fibers in well-, moderately- and poorly-differentiated OSCC's, respectively. Verhoeff's Van-Gieson showed negative staining for elastic fibers around tumor islands in different grades of OSCCs. PAS stain showed moderate staining for glycoprotein in well-differentiated OSCC and negative in moderately and poorly differentiated cases. Picrosirius red stain showed Type 1 collagen fibers in well and moderately differentiated OSCC cases and Type 3 collagen fibers in poorly differentiated cases. The observations of this study revealed altered staining reactions of the collagenous stroma and glycoproteins suggesting that tumor cells may release certain enzymes that play a role in the manipulation of ECM to enhance their own survival.

Biochemival Characterization of an Antibactrial Glycoprotein from Achatina fulica ferussac Snail Mucus Local Isolate and Their Implication on Bacterial Dental Infection

Snails crawl over a variety of potentially contaminated surfaces and their foot is the primary site of entry forpathogens, parasites and a range of opportunistic organisms, so it is a little wonder that they must have a defensivesystem to protect them. The mucus secreted on the body surfaces of mollusks is known to play crucial role inlocomotion, feeding, osmoregulation, reproduction and protection of epithelial surfaces. The snail mucus alsocontains Glycoaminoglycans (GAGs) which are complex polysaccharides that participate in the regulation ofphysiological processes through the interactions with a wide variety of proteins. GAGs, such as heparin, serve as keyto biological response modifiers, in example for acting asa a target for pathogen and parasitic factors for attachment,invasion, and immune system.For years, it has been known that the mucus secretions from snails Achatina fulica ferussac local isolate can be usedas a medication, and even empirically it is used to treat infected teeth tahat is suffered by people in rural area. Theantibacterial factor was surveyed in the aqueous extract and the mucin fraction of snail Achatina fulica ferussac, andthey exhibited positive antibacterial for Gram-positive, Escherichia coli and Gram negative, Streptococcus mutans. Inthe following study, it has been proved that an antibacterial content in the mucus was a Glycoprotein. It wascomposed of two subunits of Molecular Weight (MW) 71-73 kDa. The GelCode Glycoprotein Staining Kit detectedglycoprotein sugar moieties in polyacrylamide gel and on nitrocellulose membrane, while the glycoproteincarbohydrate estimation kit detected glycoprotein and estimated carbohydrate content. The glycoprotein contentwas 4.537 ± 0.876 for carbohydrate and 6.420 ± 1.242 for protein.Keywords : characterization, glycoprotein, Achatina fullica Ferussac snail mucus, galur Jawa, antibacterialfactor