β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.
Animals ; Aspergillus niger ; Calibration ; Cellulase/analysis* ; Chemistry, Clinical/methods* ; Dextranase/analysis* ; Enterocolitis, Necrotizing/diagnosis* ; Equipment Design ; Flavonoids/analysis* ; Glucose/analysis* ; Glucuronic Acid/analysis* ; Glucuronidase/analysis* ; Glycoside Hydrolases/analysis* ; Hydrogen-Ion Concentration ; Linear Models ; Multienzyme Complexes/analysis* ; Plants, Medicinal ; Polygalacturonase/analysis* ; Rats ; Reproducibility of Results ; beta-Galactosidase/analysis* ; beta-Glucosidase/analysis*

CD57 (synonyms: Leu-7, HNK-1) is a well-known marker of nerve elements including the conductive system of the heart, as well as natural killer cells. In lung specimens from 12 human fetuses at 10–34 weeks of gestation, we have found incidentally that segmental, subsegmental, and more peripheral arteries strongly expressed CD57. Capillaries near developing alveoli were often or sometimes positive. The CD57-positive tissue elements within intrapulmonary arteries seemed to be the endothelium, internal elastic lamina, and smooth muscle layer, which corresponded to tissue positive for a DAKO antibody reactive with smooth muscle actin we used. However, the lobar artery and pulmonary arterial trunk as well as bronchial arteries were negative. Likewise, arteries in and along any abdominal viscera, as well as the heart, thymus, and thyroid, did not express CD57. Thus, the lung-specific CD57 reactivity was not connected with either of an endodermal- or a branchial arch-origin. CD57 antigen is a sugar chain characterized by a sulfated glucuronic acid residue that is likely to exist in some glycosphingolipids. Therefore, a chemical affinity or an interaction might exist between CD57-positive arterioles and glycosphingolipids originating from alveoli, resulting in acceleration of capillary budding to make contact with the alveolar wall. CD57 might therefore be a functional marker of the developing air-blood interface that characterizes the fetal lung at the canalicular stage.
Acceleration ; Actins ; Antigens, CD57 ; Arteries* ; Arterioles ; Bronchial Arteries ; Capillaries ; Endothelium ; Fetus ; Glucuronic Acid ; Glycosphingolipids ; Heart ; Humans* ; Killer Cells, Natural ; Lung* ; Muscle, Smooth ; Pregnancy ; Thymus Gland ; Thyroid Gland ; Viscera

In the present study, a gastric retention floating system for Brucea javanica oil, composed of alginate and carrageenan, was prepared using ionotropic gelation. Parameters for floatability, drug load, encapsulation efficiency, bead morphology, in vitro release, and in vivo gastric retention were evaluated. The optimized formulation via Box-Behnken design consisted of 1.7% alginate (W/V), 1.02% carrageenan (W/V), 1.4% CaCO (W/V), and a gelling bath of pH 0.8. The alginate-carrageenan-Brucea javanica oil beads had a porous structure and exhibited up to 24 h of in vitro floatability with a load capacity of 45%-55% and an encapsulation efficiency of 70%-80%. A 6-h sustained release was observed in vitro. The beads had a prolonged gastric retention (> 60% at 6 h) in fasted rats, compared to non-floating beads (15% at 6 h), as measured by gamma scintigraphy with single-photon emission tomography/computed tomography (SPET/CT). In conclusion, the alginate-carrageenan-Brucea javanica oil system showed enhanced oil encapsulation efficiency, excellent floating and gastric retention abilities, and a favorable release behavior.
Alginates ; chemistry ; Animals ; Biological Availability ; Brucea ; chemistry ; Carrageenan ; chemistry ; Delayed-Action Preparations ; administration & dosage ; chemistry ; pharmacokinetics ; Drug Carriers ; chemistry ; Drug Delivery Systems ; methods ; Drug Evaluation, Preclinical ; Gastric Mucosa ; metabolism ; Glucuronic Acid ; chemistry ; Hexuronic Acids ; chemistry ; Microspheres ; Plant Oils ; administration & dosage ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates.AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC).On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all<0.01).Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.
Albumins ; biosynthesis ; metabolism ; Alginates ; Ammonia ; metabolism ; Animals ; Cell Aggregation ; physiology ; Cell Culture Techniques ; methods ; Cell Line, Transformed ; physiology ; Chitosan ; Diazepam ; metabolism ; Glucuronic Acid ; Hep G2 Cells ; cytology ; physiology ; Hepatocytes ; cytology ; physiology ; Hexuronic Acids ; Humans ; Liver, Artificial ; Rotation

OBJECTIVETo explore the improvement the dingle technology through the PICC catheter puncture point elbow hemostatic effect. Selection.

METHODSBetween January 2013 and December 2013, chest hospital affiliated to Shanghai jiaotong university under the guidance of ultrasound improved the Ding Gehang PICC catheter patients of 997 cases were randomly divided into three groups A, B, C, respectively, using gauze pad, calcium alginate wound dressings, calcium alginate wound dressings with hemostatic gauze pad three methods to puncture point, compare the three groups within 48 h after puncture biopsy in patients with some local bleeding, treatment times and catheter after 1 week of the maintenance costs of the catheter.

RESULTSCompared with A, B two groups, patients of group C tube after 48 hours the puncture point local oppression hemostasis effect is better than that of group A and B, the difference was statistically significant (all P < 0.05); Catheter maintenance: group C within 1 week after catheter tube after local lowest maintenance cost.

CONCLUSIONPICC for surgery after the puncture point of oppression hemostasis method choice, the effect of calcium alginate dressings hemostatic gauze pad is better than that of gauze pads and calcium alginate dressings, calcium alginate dressings and gauze pad is more effective and economic, in clinical use.

Alginates ; Bandages ; Catheters ; China ; Glucuronic Acid ; Hemorrhage ; Hemostatic Techniques ; Hexuronic Acids ; Humans ; Punctures

OBJECTIVEAlthough chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen.

METHODSChondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively.

RESULTSMMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001).

CONCLUSIONSThe study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

ADAM Proteins ; genetics ; metabolism ; Aggrecans ; genetics ; metabolism ; Alginates ; pharmacology ; Animals ; Cartilage, Articular ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; metabolism ; ultrastructure ; Collagen Type II ; genetics ; metabolism ; Female ; Glucans ; pharmacology ; Glucuronic Acid ; pharmacology ; Hexuronic Acids ; pharmacology ; Hydrogel, Polyethylene Glycol Dimethacrylate ; pharmacology ; Immunohistochemistry ; Matrix Metalloproteinase 3 ; metabolism ; Mice, Nude ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Tissue Engineering ; methods

OBJECTIVETo fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions.

METHODSWe chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis.

RESULTSCells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05).

CONCLUSIONSThe cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.

Alginates ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; physiopathology ; Cell Culture Techniques ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Glucuronic Acid ; Hexuronic Acids ; Humans ; Lung Neoplasms ; metabolism ; pathology ; physiopathology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; Printing, Three-Dimensional ; Sepharose ; Spheroids, Cellular ; pathology ; Time Factors ; Tissue Scaffolds ; Tumor Microenvironment

This study was to explore a better three-dimensional (3-D) culture method of chondrocyte. The interpenetrating network (IPN) gel beads were developed through a photo-cross linking reaction with mixed barium ions and calcium ions at the ratio of 5:5 with the methacrylic alginate (MA), which was a chemically conjugated alginate with methacrylic groups. The second generation of primary cartilage cells was encapsulated in the MA gel beads for three weeks. In the designated timing, HE stain, Alamar blue method and Scanning electron microscopic were used to determine the cartilage cells growth, proliferation and the cell distribution in the scaffolds, respectively. The expression of type II collagen was investigated by an immunohistochemistry assay and the glycosaminoglycan content was quantitatively evaluated with the spectrophotometry of 1, 9 dimethylene blue assay. Compared to the alginate control group, the deposition of glycosaminoglycan was significantly upregulated in IPN-MA gel beads with higher cell proliferation. The secretion of extracellular matrix and proliferation of chondrocyte in methacrylic alginate gel beads were higher than that in Alginate beads. Cells were able to attach, to grow well on the scaffolds under scanning electron microscopy. The result of immunohistochemistry staining of collagen type II was positive, confirming the maintenance of chondrocyte phenotype in methacrylic alginate gel beads. This study shows a great potential for three-dimensional culture of cartilage.
Alginates ; chemistry ; Barium ; chemistry ; Calcium ; chemistry ; Cartilage ; cytology ; Cations ; Cell Culture Techniques ; instrumentation ; Cells, Cultured ; Chondrocytes ; cytology ; Collagen Type II ; chemistry ; Glucuronic Acid ; chemistry ; Glycosaminoglycans ; chemistry ; Hexuronic Acids ; chemistry ; Metals ; chemistry ; Microscopy, Electron, Scanning

This study aims to explore the effects of alginate-poly ornithine-alginate (A-PLO-A) and barium alginate-poly ornithine-alginate (B-PLO-A) microcapsules as cells carriers during implantation. Mice hepatocytes coated in A-PLO-A and B-PLO-A microcapsules were implanted into rats with acute liver failure caused by intraperitoneal injection of D-galactosamine. The rat survival rate, liver cell growth, proliferation and metabolism within the microcapsules were investigated, as well as its effect on the improvement of rat acute liver failure. The influence of A-PLO-A-free microcapsules, B-PLO-A-free microcapsules, isolated liver cells, A-PLO-A microcapsule-coated and B-PLO-A microcapsule-coated liver cells was studied. It was found that the chemical-free microcapsules showed no positive effect on the rats with liver failures, with a death rate of 100% in both groups 3 days after the implantation. The ALT, AST and ALB levels were all improved in the isolated liver cell group, the A-PLO-A microcapsule-coated and the B-PLO-A microcapsule-coated groups. The survival rate of both microcapsule-coated liver cell groups was significantly higher than that of the chemical-free microcapsule group and the isolated liver cells group. The microcapsules were retrieved after 4 weeks' implantation, which were observed to be smooth with no cells attaching to the surface. No apparent fibrosis was observed. This research demonstrated the physical stability and the biocompatibility of the PLO-based alginate microcapsules and therefore they could be used as liver cell carriers during implantation.
Alginates ; administration & dosage ; Animals ; Glucuronic Acid ; administration & dosage ; Hepatocytes ; transplantation ; Hexuronic Acids ; administration & dosage ; Liver Failure ; therapy ; Mice ; Ornithine ; administration & dosage ; Rats

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
Animals ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2C9 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Drug Interactions ; Glucuronosyltransferase ; metabolism ; Humans ; Ligusticum ; chemistry ; Microsomes, Liver ; enzymology ; NADP ; metabolism ; pharmacology ; Pyrazines ; metabolism ; pharmacokinetics ; Rats ; Uridine Diphosphate Glucuronic Acid ; metabolism ; pharmacology