Periodic acid-Schiff Reagent Staining Research Articles

N-acetylcysteine combined with insulin therapy can reduce myocardial injury induced by type 1 diabetes through the endoplasmic reticulum pathway

With the development of Type 1 diabetes mellitus (T1DM), various complications can be caused. Hyperglycemia affects the microenvironment of cardiomyocytes, changes endoplasmic reticulum homeostasis, triggers unfolding protein response and eventually promotes myocardial apoptosis. However, insulin therapy alone cannot effectively combat the complications caused by T1DM. Forty adult beagles were randomly divided into five groups: control group, diabetes mellitus group, insulin group, insulin combined with NAC group, and NAC group. 24-hour blood glucose, 120-day blood glucose, 120-day body weight, and serum FMN content were observed, furthermore, hematoxylin-eosin staining, Periodic acid Schiff reagent staining, and Sirius red staining of the myocardium were evaluated. The protein expressions of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase 3, Bcl2, and Bax were detected. Results of the pathological section of myocardial tissue indicated that insulin combined with NAC therapy could improve myocardial pathological injury and glycogen deposition. Additionally, insulin combined with NAC therapy down-regulates the expression of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase3, and Bax. These findings suggest that NAC has a phylactic effect on myocardial injury in beagles with T1DM, and the mechanism may be related to the improvement of endoplasmic reticulum stress-induced apoptosis.

A trimeric glycosylated GH45 cellulase from the red abalone (Haliotis rufescens) exhibits endo and exoactivity.

The red abalone (Haliotis rufescens) represents North America's most important aquaculture species. Its hepatopancreas is rich in cellulases and other polysaccharide-degrading enzymes, which provide it the remarkable ability to digest cellulose-rich macroalgae; nevertheless, its cellulolytic systems are poorly explored. This manuscript describes some functional and structural properties of an endogenous trimeric glycosylated endoglucanase from H. rufescens. The purified enzyme showed a molecular mass of 23.4 kDa determined by MALDI-TOF mass spectrometry, which behaved as a homotrimer in gel filtration chromatography and zymograms. According to the periodic acid-Schiff reagent staining, detecting sugar moieties in SDS-PAGE gel confirmed that abalone cellulase is a glycoprotein. Hydrolysis of cello-oligosaccharides and p-nitrophenyl-β-D-glucopyranosides confirmed its endo/exoactivity. A maximum enzyme activity toward 0.5% (w/v) carboxymethylcellulose of 53.9 ± 1.0 U/mg was achieved at 45°C and pH 6.0. We elucidated the abalone cellulase primary structure using proteases and mass spectrometry methods. Based on these results and using a bioinformatic approach, we identified the gene encoding this enzyme and deduced its full-length amino acid sequence; the mature protein comprised 177 residues with a calculated molecular mass of 19.1 kDa and, according to sequence similarity, it was classified into the glycosyl-hydrolase family 45 subfamily B. An AlphaFold theoretical model and docking simulations with cellopentaose confirmed that abalone cellulase is a β-sheet rich protein, as also observed by circular dichroism experiments, with conserved catalytic residues: Asp26, Asn109, and Asp134. Interestingly, the AlphaFold-Multimer analysis indicated a trimeric assembly for abalone cellulase, which supported our experimental findings. The discovery and characterization of these enzymes may contribute to developing efficient cellulose bioconversion processes for biofuels and sustainable bioproducts.

Secreted Ly-6/uPAR-related protein-1 (SLURP1) is a pro-differentiation factor that stalls G1-S transition during corneal epithelial cell cycle progression

PurposePreviously we demonstrated that the secreted Ly-6/uPAR related protein-1 (SLURP1), abundantly expressed in the corneal epithelium (CE) and secreted into the tear fluid, serves as an anti-inflammatory and anti-angiogenic molecule. Here we describe the Slurp1-null (Slurp1X−/−) mouse corneal phenotype for the first time. MethodsWe compared the 10-week-old wild type (WT) and Slurp1X−/− mouse corneal (i) histology by hematoxylin-eosin and periodic acid-Schiff's reagent staining, (ii) cell proliferation by immunostaining for Ki67, (iii) cell adhesion molecules by immunostaining for desmosomal and tight junction proteins, (iv) barrier function by fluorescein staining and (v) wound-healing by epithelial debridement. Effect of SLURP1 on cell cycle was quantified in human corneal limbal epithelial (HCLE) cells engineered to express SLURP1 (HCLE-SLURP1). ResultsWT and Slurp1X−/− corneal histology was largely comparable, other than a few loosely attached superficial cells in Slurp1X−/− corneas. Compared with the WT, Slurp1X−/− corneas displayed (i) increase in Ki67+ cells, (ii) altered expression and/or localization of tight junction proteins Tjp1 and Pard3, and desmosomal Dsp, (iii) increased superficial fragility and (iv) slower CE wound healing. HCLE-SLURP1 cells displayed (i) decrease in Ki67+ cells, (ii) increased cell number doubling time, (iii) stalling in G1-S phase transition during cell cycle, and (iv) downregulation of cyclins CCNE and CCND1/D2, cyclin-dependent kinases CDK4 and CDK6, and upregulation of CDK inhibitor p15/CDKN2B. ConclusionsCollectively, these results elucidate that Slurp1X−/− CE cell homeostasis is altered and suggest that SLURP1 is a pro-differentiation factor that stalls G1-S transition during cell cycle progression by downregulating cyclins and upregulating p15/CDKN2B.

Comparison of the Effects of Intraperitoneal Injection with Carbon Tetrachloride on Acute Liver Toxicity in Male and Female Kunming Mice.

BackgroundAcute chemical liver injury needs to be further explored. The present study aimed to compare the effects of intraperitoneal injection with carbon tetrachloride on acute liver toxicity after 24 h in male and female Kunming mice.Material/MethodsIn this study, female and male mice were simultaneously divided into 3 different groups. Each group was treated differently, and after 24 h, blood samples were collected to check for changes in the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were used to assess liver toxicity. Liver samples were used for hematoxylin-eosin staining, and periodic acid Schiff reagent staining was performed to detect the pathological changes of each group. The expression level of biomarker molecules in liver cells was also systematically analyzed.ResultsOur results showed that, compared with male mice, female mice showed more serious damage: reduced glycogen and higher degree of necrosis, and the levels of heatshock protein 27 (HSP27), heat-shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA) and B cell lymphoma/lewkmia-2 (Bcl-2) were significantly lower than in the male group (P<0.05 or P<0.01), while the results of Bcl-2-associated X protein (Bax), cysteinyl aspartate specific proteinase 3 (Caspase3), and cytochrome P450 2E1 (CYP2E1) were the opposite (P<0.05 or P<0.01).ConclusionsThe findings from this study showed that, compared with male mice, at 24 h after CCl4 toxicity, female mice showed more severe changes of hepatocyte necrosis and PAS-positivity, with significantly reduced expression of HSP27, HSP70, PCNA, and Bcl-2, and significantly increased expression of Bax, caspase-3, and CYP2E1.

Application of fungal fluorescent staining in oral candidiasis: diagnostic analysis of 228 specimens

BackgroundSeveral conventional methods, including fungal culture and periodic acid-Schiff (PAS) reagent staining, have been used to diagnose oral candidiasis. The aim of this study was to evaluate the efficacy of a novel method, fungal fluorescent staining, in relation to conventional protocols in the diagnosis of oral candidiasis.MethodsWe collected 106 oral swabs and 122 oral biopsy tissues from patients highly suspected with oral candidiasis. We applied fungal culture and periodic acid-Schiff reagent staining as the gold standard diagnostic tools. The efficacy of these methods in determining the presence of Candida was compared with that of fluorescent staining.ResultsIn the majority of specimens subjected to fluorescent staining, fungal organisms were distinguished by blue fluorescence surrounding their tubular or annular shapes. The sensitivity, specificity, Youden index, positive predictive value and negative predictive value of the fluorescent staining method were 82.7, 93.5, 76.7, 96.8 and 69.1% in oral swabs and 90.0, 92.9, 82.9, 96.0 and 82.9% in oral biopsy tissues, respectively.ConclusionsFungal fluorescent staining represents a rapid method for detection of Candida, supporting its potential utility as an effective early diagnostic tool for oral candidiasis.

Antidiabetic effects of different polysaccharide fractions from Artemisia sphaerocephala Krasch seeds in db/db mice

Artemisia sphaerocephala Krasch polysaccharide (ASKP) has previously been reported to ameliorate the hyperglycemia and oxidative stress in type 2 diabetes mellitus and was separated into two fractions of 60P and 60S. To determine the major functional fraction in ASKP, the antidiabetic effects of ASKP and its two fractions were investigated in diabetic db/db mice. As a result, 60P showed the highest and 60S showed the lowest capability in many aspects: decreasing the fasting blood glucose, glycated serum protein, and the insulin resistance in db/db mice; reversing the dyslipidemia caused by diabetes as indicated by the decreased levels of total cholesterol, triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, and non-esterified fatty acid; as well as exerting antioxidant activities (increases in catalase, superoxide dismutase, and glutathione peroxidase, and a decrease in malondialdehyde) and hepatoprotective activities (increases in total protein and albumin, decreases in alanine aminotransferase and aspartate aminotransferase) in the liver of db/db mice. Liver histopathological observation, using both hematoxylin-eosin staining and periodic acid-Schiff reagent staining, indicated that all polysaccharide fractions could ameliorate the deterioration of hepatic lesions and increase the hepatic glycogen storage in db/db mice. Furthermore, analysis of relative mRNA expression levels suggested that all polysaccharide fractions promoted the glycogen synthesis by up-regulating the expressions of peroxisome proliferator-activated receptor-γ, glucose transporter-4, phosphoinositide 3-kinase, protein kinase B, and glucokinase and inhibited gluconeogenesis via down-regulating the expression of phosphoenopyruvate carboxykinase. Overall, 60P showed as the major antidiabetic fraction in ASKP.

Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis.

Glycogen accumulation occurs in beta cells of diabetic patients and has been proposed to partly mediate glucotoxicity-induced beta cell dysfunction. However, the role of glycogen metabolism in beta cell function and its contribution to diabetes pathophysiology remain poorly understood. We investigated the function of beta cell glycogen by studying glucose homeostasis in mice with (1) defective glycogen synthesis in the pancreas; and (2) excessive glycogen accumulation in beta cells. Conditional deletion of the Gys1 gene and overexpression of protein targeting to glycogen (PTG) was accomplished by Cre-lox recombination using pancreas-specific Cre lines. Glucose homeostasis was assessed by determining fasting glycaemia, insulinaemia and glucose tolerance. Beta cell mass was determined by morphometry. Glycogen was detected histologically by periodic acid-Schiff's reagent staining. Isolated islets were used for the determination of glycogen and insulin content, insulin secretion, immunoblots and gene expression assays. Gys1 knockout (Gys1 (KO)) mice did not exhibit differences in glucose tolerance or basal glycaemia and insulinaemia relative to controls. Insulin secretion and gene expression in isolated islets was also indistinguishable between Gys1 (KO) and controls. Conversely, despite effective glycogen overaccumulation in islets, mice with PTG overexpression (PTG(OE)) presented similar glucose tolerance to controls. However, under fasting conditions they exhibited lower glycaemia and higher insulinaemia. Importantly, neither young nor aged PTG(OE) mice showed differences in beta cell mass relative to age-matched controls. Finally, a high-fat diet did not reveal a beta cell-autonomous phenotype in either model. Glycogen metabolism is not required for the maintenance of beta cell function. Glycogen accumulation in beta cells alone is not sufficient to trigger the dysfunction or loss of these cells, or progression to diabetes.

Involvement of Endoplasmic Reticulum Stress in Uremic Cardiomyopathy: Protective Effects of Tauroursodeoxycholic Acid

Background/Aims: Uremic cardiomyopathy (UCM) is a complication in chronic kidney disease. We investigated if endoplasmic reticulum stress (ERS) is involved in UCM, and determined the efficacy of tauroursodeoxycholic acid (TUDCA) in UCM prevention. Methods: Mice were divided randomly into three groups: sham (saline, i.p), 5/6 nephrectomized (Nx) (saline, i.p) and Nx+TUDCA (250 mg/kg/day, i.p.). Renal function was assessed by measuring serum creatinine, blood urea nitrogen and by periodic acid-Schiff reagent staining. Histologic examination of cardiac fibrosis and apoptosis was determined by Masson's trichrome and TUNEL assay. Cardiac function was evaluated by echocardiography. Fibrotic factors (transforming growth factor-β, fibronectin, collagen I/IV) were evaluated by real-time PCR. ERS-related proteins were measured by western blotting. Results: Impaired renal function and cardiac dysfunction were shown in 5/6 nephrectomy mice but were improved significantly by TUDCA. 5/6 nephrectomy mice exhibited marked cardiomyocyte apoptosis, cardiac fibrosis and elevated pro-fibrotic factors. ERS markers (GRP78, GRP94, P-PERK, P-eIF2a) and ERS-induced apoptosis pathways (activation of CHOP and caspase-12) were increased significantly in 5/6 nephrectomy mice, and TUDCA treatment blunted these changes. Conclusions: ERS has a key role in UCM, and the cardioprotective role of TUDCA is related to inhibition of ERS-induced apoptosis by inhibition of CHOP and caspase-12 pathways.

A primary study of L-carnitine protective effect on corneal and conjunctival epithelium of mouse dry eye model induced by hyperosmolar saline

To evaluate protective effects of L-carnitine on corneal and conjunctival epithelium of mouse dry eye model induced by hyperosmolar saline. Sixty female BALB/c mice at the age of 6 - 8 weeks were randomly divided into three groups (20 in each): Hyperosmolar saline group (HO), Hyperosmolar and Isosmotic saline group (HO + IO), as well as Hyperosmolar saline and 1% L-carnitine group (HO + 1%LCA). The HO group was treated with 500 mOsmol/L sodium chloride solution; the HO + IO group with 308 mOsmol/L sodium chloride solution first and 500 mOsmol/L sodium chloride solution 30 minutes later; and the HO + 1%LCA group with 1%L-carnitine eye drop first and 500 mOsmol/L sodium chloride solution 30 minutes later. Alternately, 5 times a day for 28 days. Corneal fluorescein staining, corneal epithelial hematoxylin-eosin staining and thickness measurement, conjunctival epithelial periodic acid-schiff reagent staining and goblet cell counting were conducted on 0, 7th, 14th and 28th days, respectively. On 28th day, corneal surface was inspected by scaning electron microscopy and tear osmolarity was measured. In HO or HO + IO group, compared with HO + 1%LCA group, the number of goblet cells was reduced, the score of corneal fluorescein staining and corneal epithelial thickness was elevated: on 7th day, there was difference noticed in the corneal epithelial thickness [(27.7 ± 1.1), (26.5 ± 1.4), (25.1 ± 1.0) µm, P < 0.01] and the number of goblet cell [(11.9 ± 1.1), (12.1 ± 0.9), (13.3 ± 0.9), P < 0.01] in 3 groups; on 14th day, there was evident change in the corneal fluorescein staining area [(3.2 ± 0.9), (2.9 ± 0.7), (1.7 ± 0.8), χ(2) = 11.465, P < 0.01] in 3 groups. On 28th day, Increased tear osmolarity and lowered microvilli on cornea were examined in HO [(327.37 ± 4.20) mOsmol/L] or HO + IO [(324.36 ± 5.72) mOsmol/L] group compared with HO + 1%LCA [(308.29 ± 5.72) mOsmol/L] group (P < 0.01). L-carnitine had protective effect on corneal and conjunctival epithelium of mouse dry eye model induced by hyperosmolar saline, and should be studied further.

Expression of vascular endothelial growth factor by granulated metrial gland cells in pregnant murine uteri.

Granulated metrial gland (GMG) cells are a characteristic uterine component belonging to a natural killer cell lineage. This study is aimed at revealing their kinetic and spatial relationship with vascular growth during pregnancy and the expression of vascular endothelial growth factor (VEGF). GMG cells and blood vessels were identified by periodic-acid-Schiff-reagent (PAS)-stained granules and positive staining for factor-VIII-related antigen, respectively. GMG cells were widely distributed in the decidua and metrial gland and showed a numerical increase with a peak at day 13 in parallel with the increase of vascular density. Preceding the maximal vascular development at day 13, microvessels with a narrow lumen representative of neovascularization prevailed at days 7-9, and the VEGF content in the decidua/metrial gland was significantly elevated at days 7-13 concurrently with mRNA expression. By immunolight microscopy combined with PAS staining, GMG cells with PAS-stained granules were positive for VEGF. Immunoelectron microscopy demonstrated that immunoreactions were diffuse in the cytoplasm but not localized in the granules. In contrast, fibroblast-like stromal cells were negative. These data indicate that GMG cells express VEGF and may play inducing roles in uterine neovascularization during pregnancy.

Detection of distinct species in purified human respiratory mucin using monoclonal antibodies

The purpose of this investigation was to demonstrate the presence of different species (subpopulations) in the purified human tracheobronchial mucin (HTM-1). Mucin was highly purified from sputum specimens collected from a cystic fibrosis (CF) patient using a protocol involving sequential chromatography on Bio-Gel A-5m and hydroxylapatite columns. SDS-composite gel electrophoresis followed by periodic acid-Schiff's reagent staining was unable to detect mucin species. However, using enzyme-linked immunoelectrotransfer blot (EITB) method and polyclonal antibodies raised against HTM-1, at least four different migrating mucin species were detected. Further immunological characterization of these mucin species was carried out using a library of 16 monoclonal antibodies (MAbs) developed against the purified mucin. Nine MAbs belonged to the IgM class, two MAbs were IgG1, one IgG2a and remaining four were of the IgG 3 subclass. Periodate oxidation of the mucin antigen was used to establish the nature of the mucin epitopes recognized by the MAbs. 11 MAbs recognized carbohydrate epitopes in the mucin molecule that were sensitive to periodate, while five MAbs reacted with periodate resistant carbohydrate epitopes or the protein portion of the mucin molecule. Enzyme-linked immunoelectrotransfer blot analysis of the MAbs against HTM-1 showed the presence of at least three distinct mucin species. Chromatography of the mucin on immunoaffinity columns (MAbs H(13.3), M(33.3) and CCK 061 conjugated to CNBr-activated Sepharose 4B), followed by ELISA and EITB analyses, established the mucin species recognized by the antibodies. These experiments further indicated that both unique and shared epitopes were present in the mucin species. These monoclonal antibodies may provide a promising approach to differentiate the secretory products of the tracheobronchial tree.

Extracellular phytase (E.C. 3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization.

Extracellular phytase from Aspergillus ficuum, a glycoprotein, was purified to homogeneity in 3 column chromatographic steps using ion exchange and chromatofocusing. Results of gel filtration chromatography and SDS-polyacrylamide gel electrophoresis indicated the approximate molecular weight of the native protein to be 85-100-KDa. On the basis of a molecular weight of 85-KDa, the molar extinction coefficient of the enzyme at 280 nm was estimated to be 1.2 X 10(4) M-1 cm-1. The isoelectric point of the enzyme, as deduced by chromatofocusing, was about 4.5. The purified enzyme is remarkably stable at 0 degree C. Thermal inactivation studies have shown that the enzyme retained 40% of its activity after being subjected to 68 degrees C for 10 minutes, and the enzyme exhibited a broad temperature optimum with maximum catalytic activity at 58 degrees C. The Km of the enzyme for phytate and p-nitrophenylphosphate is about 40 uM and 265 uM, respectively, with an estimated turnover number of the enzyme for phytate of 220 per sec. Enzymatic deglycosylation of phytase by Endoglycosidase H lowered the molecular weight of native enzyme from 85-100-KDa to about 76-KDa; the digested phytase still retained some carbohydrate as judged by positive periodic acid-Schiff reagent staining of the electrophoresed protein. Immunoblotting of the phytase with monoclonal antibody 7H10 raised against purified native enzyme recognized not only native but also partially deglycosylated protein.

Aggregation of surface mine soil by interaction between VAM fungi and lignin degradation products of lespedeza

The external mycelium of a vesicular-arbuscular mycorrhizal (VAM) fungus was effective in aggregating a sandy loam minesoil. The polysaccharide nature of the soil binding agent on hyphal surfaces and on the surfaces of sand particles in contact with the hyphae within the aggregate was demonstrated with the periodic acid-Schiff reagent staining reaction. A possible stabilizing mechanism for macroaggregates was proposed that involves a coupling reaction between glucosamines in the hyphal walls of the fungus with phenolic compounds released during lignin degradation of sericea lespedeza root tissue.

Caulobacter crescentus pilin. Purification, chemical characterization, and NH2-terminal amino acid sequence of a structural protein regulated during development.

The pili of Caulobacter crescentus are assembled during swarmer cell development at the differentiating cell pole. Under specific growth conditions, it was found that C. crescentus CB15 will produce insoluble pigmented granules which entrap pili lost during cell growth along with other extracellular proteins. This provided a strategy for protein purification. Pilin was purified from these granules by gel filtration in the presence of high levels of detergent. A protein of the appropriate molecular weight for pilin was isolated by this procedure and demonstrated to be pilin by specific labeling of the intact pilus with ferritin-coupled antibodies. CB15 pilin has an apparent molecular weight of 8,000, is rich in hydrophobic amino acids (greater than 70%), and has a broad isoelectric focusing range centered about a pI of 6.6. Periodic acid-Schiff reagent staining did not demonstrate carbohydrate modification of the monomer. The pilins of both CB15 and a related strain CB13 cross-react with anti-CB15 pilin antibody. Both strains also demonstrate similar RNA bacteriophage sensitivity, although there were significant differences in the amino acid composition, molecular weight, and other physical properties of the pilins from these two related strains. The NH2-terminal amino acid sequence of about 40% of the CB15 pilin molecular has been determined and bears some resemblance to that of some common pili; however, the sequences are not homologous and there is no indications of an unusual NH2-terminal amino acid in Caulobacter pilin.

Additional platelet membrane glycoprotein abnormalities in glanzmann's thrombasthenia: A comparison with normals by high resolution two-dimensional polyacrylamide gel electrophoresis

The platelets from sixteen patients with Glanzmann's thrombasthenia have been analysed by isoelectric focusing and gradient sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique and compared with normals. As well as the previously described absence or deficiency of glycoproteins IIb and IIIa and a marked reduction in fibrinogen levels there were changes in the isoelectric point of glycoproteins Ib and IIIb and increased periodic acid-Schiff's reagent staining of some other glycoproteins. These differences imply that in Glanzmann's thrombasthenia there is more general perturbation of platelet membrane glycoproteins than previously suspected.

Distribution of glycogen in prefusion human palatal epithelium

Various stages of embryonic human secondary palatal development were examined for the presence of epithelial glycogen. Utilizing periodic acid-Schiff's reagent staining of thick plastic sections and osmium ferrocyanide enhancement of thin sections, dramatic changes in epithelial glycogen distribution were noted during palatogenesis. Prior to fusion, the epithelium destined to adhere in the midline exhibited a marked diminution of glycogen in the superficial cell layer. This cell layer was composed of slender dense cells and cuboidal cells undergoing lysis. Adjacent nonfusing epithelium was markedly different and contained large glycogen reserves in its superficial cell layer. Glycogen may play a role either as precursor for specific adhesive macromolecules or as a physical agent capable under the influence of appropriate enzymes of causing cell lysis.

Molecular transport: I. In vitro studies of isolated glycoprotein subunits of the amino acid transport system of Neurospora crassa conidia

Neurospora crassa conidia have constitutive transport systems for neutral and basic amino acids. KCl-extracted conidia show reduced l-arginine transport. KCl extracts contain several molecular species, some of which will bind l-amino acids. Purification of these extracts by affinity chromatography yields two glycoprotein species with a relatively high affinity for l-arginine. The chromatographic disappearance of one glycoprotein species is associated with a single gene mutation resulting in reduction of l-arginine transport in vivo. Disappearance of the second glycoprotein species from the chromatography profile is associated with modification of either of two, unlinked transport loci. Initial biochemical characterization of both molecular species indicate a weight composition of approximately 50% carbohydrate and 50% protein. Disc electrophoresis of the glycoprotein species associated with the single gene transport mutation yields a single band of both amido black and periodic acid-Schiff reagent staining material.