Genomic DNA and cDNA sequences of lactase from Aspergillus candidus were cloned. Sequences analysis revealed that the genomic DNA was 3458 bp containing eight introns, cDNA was 3015 bp and encoding a polypeptide of 1005 amino acid residues. Signal peptide was 19 amino acid residues, eleven potential N-glycosylation sites were assumed. Comparing the gene cDNA and amino acid sequences with other lactase sequences from various sources, it showed a very low homology with most of other sequences. Although the gene had a higher homology to Aspergillus oryzae lactase sequence, characterization of both enzymes exhibited obvious difference. The gene from Aspergillus candidus was a promising new gene for food industry.
Amino Acid Sequence ; Aspergillus ; enzymology ; genetics ; Cloning, Molecular ; Lactase ; Molecular Sequence Data ; Sequence Homology ; beta-Galactosidase ; chemistry ; genetics

OBJECTIVETo study the content of phytoestrogen in dissimilarity herbs.

METHODThe activity of phytoestrogen in heat-clearing drugs, drugs for relieving exterior syndrome, diuretic, anastaltics, tonics and astringents were detected based on the recombinant yeast cell (W303-1A/hER-ERE-Lac Z). The estrogenic activity in traditional Chinese materia medica were assayed quantitatively by determining the expression of beta-galactosidase.

RESULTThe phytoestrogen concentration (6.35 x 10(-3) nmol x g(-1) E2 equivalent) in heat-clearing drugs was the highest while that in anastaltic and tonic drugs was the lowest, which was less than the detected limit.

CONCLUSIONCompared with the other traditional Chinese materia medica, the content of phytoestrogen, which can bind to estrogen receptor, in giant knotweed rhizome, forsythia suspense, ash bark, baical skullcap root and ophiopogonis tuber were higher.

Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Phytoestrogens ; analysis ; metabolism ; Plants, Medicinal ; chemistry ; Receptors, Estrogen ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; chemistry ; cytology ; drug effects ; genetics ; beta-Galactosidase ; analysis

The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic bacteria and the production of thermo-stable enzymes, which pose great threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic bacteria and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an effective strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic bacteria from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activity was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activity was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70, 80, or 90 °C, and proteases appeared to be more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major concern to the dairy industry. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Animals ; Bacteria/genetics* ; Bacterial Proteins/chemistry* ; Biofilms ; Cold Temperature ; Dairy Products ; Endopeptidases/chemistry* ; Enzyme Stability ; Food Microbiology ; Hot Temperature ; Lipase/chemistry* ; Milk/microbiology* ; Peptide Hydrolases/chemistry* ; Phospholipases/chemistry* ; RNA, Ribosomal, 16S/genetics* ; Raw Foods/microbiology* ; beta-Galactosidase/chemistry*

OBJECTIVETo screen enhancer-like sequences from Escherichia coli strain C600 genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression.

METHODSEnhancer-like element from Escherichia coli strain C600 genome was obtained by using the chloramphenicol acetyl-transferase (CAT) gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence from Escherichia coli strain C600 was constructed. Interferon was expressed and assayed.

RESULTSAn enhancer-like sequences with distance and orientation independence property were screened and named 3A. Quantification test showed that the direct and reverse orientation of 3A could increase the activity of beta-galactosidase with 7.11 and 2.93 times. The enhancing activity of the element was on transcription level. An expression vector harboring the prokaryotic enhancer-like sequence 3P3 which was enhancing function region of sequence 3A was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 3.7 times in comparison with the original expression plasmid.

CONCLUSION3A enhancer-like sequence was screened from Escherichia coli strain C600 genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.

Enhancer Elements, Genetic ; genetics ; Escherichia coli ; genetics ; Gene Expression ; drug effects ; genetics ; Genes, Reporter ; Genetic Vectors ; chemistry ; Interferons ; chemistry ; genetics ; metabolism ; Prokaryotic Cells ; Sequence Homology, Nucleic Acid ; beta-Galactosidase ; genetics

OBJECTIVETo develop an objective bioassay for quantitative detection of HIV-induced cell-cell fusion for screening HIV entry inhibitors.

METHODSHL2/3 cells expressing HIV envelope proteins gp120/gp41, Tat, and other HIV proteins were co-cultured with HeLa-CD4-LTR-beta-gal cells expressing CD4 receptor and HIV LTR triggered reporter gene beta-galactosidase. The enzyme activities of beta-galactosidase were detected by a chromogenic substrate, chlorophenol red-beta-galactopyranoside (CPRG). Specific HIV entry inhibitors were used to validate the established detecting method.

RESULTSNo syncytium was formed by mixing HL2/3 and HeLa-CD4-LTR-beta-gal cells. However, the membrane could be fused and the Tat expressed by HL2/3 cells could bind to HIV LTR on HeLa-CD4-LTR-beta-gal cells and trigger the expression of beta-galactosidase. CPRG allowed quantitative and sensitive detection of the activity of beta-galactosidase. Further studies showed that HIV entry inhibitors could inhibit the activity of beta-galactosidase in a dose-dependent manner.

CONCLUSIONWe have developed a simple, cheap, objective and quantitative non-infectious cell-cell fusion bioassay that can be used to screen for anti-HIV agents targeting the virus entry from natural and synthetic compound libraries.

Biological Assay ; Cell Fusion ; Cell Line ; Coculture Techniques ; Drug Evaluation, Preclinical ; methods ; HIV Envelope Protein gp120 ; metabolism ; HIV Envelope Protein gp41 ; metabolism ; HIV Fusion Inhibitors ; chemistry ; pharmacology ; Humans ; beta-Galactosidase ; metabolism

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*

OBJECTIVETo observe the time-effect relation of extracts from ginseng, notoginseng and chuanxiong on angiotensin II (Ang II)-induced senescence of vascular endothelial cells and explore the feature of Chinese medicine against vascular diseases.

METHODSHuman umbilical vein endothelial cells (HUVECs) cultured in vitro were stimulated with 10(-6) mol/L AngII to induce cell senescence, which were divided into 4 groups, the blank control group, the Ang II model group, the extracts group and the telmisartan group. The β-gal was used to identify senescence of cells, the cell counting kit-8 method was applied to assess the cell viability, the cell function was examined with the level of endothelial nitric oxide synthase (eNOS) and the flow cytometry was used for analyzing the cell cycle changes.

RESULTSCompared with the control cells, the cells positive for &beta;-gal staining was significantly increased in the Ang II model group, and showed cell cycle arrest at G0/G1 phase with decreased S and G2/M phase cell percentage, eNOS expression and cell viability (P<0.05). The extracts and telmisartan treatment of Ang II-induced cells resulted in decreased &beta;-gal positive cells with a reduction in G0/G1 phase cells and an increasing in S, G2/M phase cells and eNOS expression (P<0.05). At 24 h, the extracts were more effective than telmisartan (P<0.05); while telmisartan was more effective at 48 h (P<0.05).

CONCLUSIONExtracts from ginseng, notoginseng and chuanxiong can delay Ang II-induced aging of HUVECs and may play an important role in early senescence.

Cell Cycle Checkpoints ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cellular Senescence ; drug effects ; Down-Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Panax notoginseng ; chemistry ; Plant Extracts ; pharmacology ; Time Factors ; beta-Galactosidase ; metabolism

OBJECTIVEThe effect of angelica sinensis polysaccharides (ASP) on the production of reactive oxygen specie (ROS), the capability of total anti-oxidant (T-AOC), and the expression of p16 in mRNA level in mice hematopoietic stem cells (HSCs) were observed to explore the underlying mechanism that ASP delay aging of HSCs in vivo.

METHODC57BL/6J mice were randomly divided into normal group, aging group, and the above groups treated with ASP. Mice were uniformly explored in X-ray (3.0 Gy/8 F) to erect model of aging. Normal and aging ASP intervention groups mice were treated with ASP by intragastric administration, while normal and aging groups were treated with equal-volume NS during X-ray irradiation. Mice HSCs were isolated by magnetic cell sorting and cultured in vitro. Senescence-associated beta-galactosidase (SA-beta-Gal) staining was used to detect aging HSCs. Cell cycles analysis and CFU-Mix cultivation were used to evaluate the capability of self-renewing and colony forming in HSCs. The production of ROS in HSCs was evaluated by flow cytometry analysis and immunofluorescence assess, respectively. T-AOC was detected by chemical colorimetric method. The expression of p16 was determined by real-time quantitative PCR (qRT-PCR).

RESULTExogenous X-ray irradiation induced HSCs aging was compared with normal group without irradiation. Biological feature of HSCs in aging group with X-ray irradiation as follows: The percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS were significantly increased , the expression of p16 in mRNA level was also upregulated. The capacility of colony forming and T-AOC in HSCs were decreased. ASP could significantly decrease the percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS in HSCs, and downregulate the expression of p16 in mRNA level in HSCs contrast to aging group without ASP treatment. In addition, ASP could remarkably increase T-AOC and the capacility of colony forming in HSCs compared with aging group without ASP treatment.

CONCLUSIONX-ray (3.0 Gy/8 F) could induce mice HSCs aging. ASP could delay senescence HSCs aging which maybe partly ascribed to the inhibition of oxidative damage and the downregulation of p16 mRNA expression.

Aging ; drug effects ; radiation effects ; Angelica sinensis ; chemistry ; Animals ; Cell Cycle ; drug effects ; radiation effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; radiation effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Female ; Flow Cytometry ; Gene Expression ; drug effects ; radiation effects ; Hematopoietic Stem Cells ; drug effects ; metabolism ; radiation effects ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; drug effects ; radiation effects ; Polysaccharides ; pharmacology ; Random Allocation ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; X-Rays ; beta-Galactosidase ; metabolism