To explore the effect of geniposidic acid (GPA) on the signal pathway of small heterodimer dimer receptor (SHP) and liver receptor homologue 1 (LRH-1) in cholestasis rats induced by alpha-naphthalene isothiocyanate (ANIT).
 Methods: Fifty SD rats were randomly divided into five groups: a blank group, an ANIT group, an ANIT+GPA (100 mg/kg) group, an ANIT+GPA (50 mg/kg) group, and an ANIT+GPA (25 mg/kg) group (n=10 in each group). The GPA were intragastrically given to rats for 10 days, and the control group and the ANIT group were given normal saline. At the eighth day of administration, all rats except the blank group were given 65 mg/kg ANIT once until the tenth day. After the last administration, serum total cholesterol (TC), triglyceride (TG) and total bile acids (TBA) were measured. The primary hepatocytes (RPH) were isolated from normal rats and cultured. The cells were divided into a blank group, an ANIT (40 μmol/L) group, an ANIT (40 μmol/L)+GPA (4.00 mmol/L) group (A4.00G group), an ANIT (40 μmol/L)+GPA (1.00 mmol/L) group (A1.00G group), and an ANIT (40 μmol/L)+GPA (0.25 mmol/L) group (A0.25G group). The mRNA transcription levels of SHP and cholesterol 7 alpha hydroxylase (CYP7A1) in RPH were detected by real-time-PCR, and the protein levels of SHP and CYP7a1 were detected by Western blotting. In the LRH-1 silence experiment, the RPH were divided into a blank group, a negative transfection group, a siRNA-LRH group (ZR group), a siRNA-LRH+GPA (4.00 mmol/L) group (ZR4.00G group), a siRNA-LRH+GPA (1.00 mmol/L) group (ZR1.00G group) and a siRNA-LRH+GPA (0.25 mmol/L) group (ZR0.25G group). The protein and mRNA levels of SHP, CYP7a1, LRH-1 were detected. In the over-expression experiment, the RPH were also divided into a blank group, a negative transfection group, a LRH-1 over-expression plasmid group (OE group), a LRH-1 over-expression plasmid+GPA (4.00 mmol/L) group (OE4.00G group), a LRH-1 over-expression plasmid+GPA (1.00 mmol/L) group (OE1.00G group), and a LRH-1 over-expression plasmid+GPA (0.25 mmol/L) group (OE0.25G group). The protein and mRNA levels of SHP, CYP7a1 and LRH-1 were detected.
 Results: Compared with the blank control group, TC and TBA were significantly increased (both P<0.01) in the ANIT group, but there was no difference in TG; compared with the ANIT group, the contents of TC and TBA in the AG100 and AG50 groups were significantly reduced (all P<0.01). Compared with the blank control group, the proteins and mRNA levels of SHP were significantly decreased (P<0.01), while CYP7a1 were dramatically increased (P<0.01) in the ANIT group; compared with the ANIT group, the proteins and mRNA levels of SHP in the A4.00G group and the A1.00G group were significantly increased (both P<0.01), while the levels of CYP7a1 proteins and mRNA levels were evidently decreased in the A4.00G and A1.00G groups (both P<0.01). Compared with the negative transfection group, the proteins and mRNA levels of CYP7a1 and LRH-1 were dramatically restrained (all P<0.01), while there was no change in SHP in the ZR group; compared with the ZR group, the proteins and mRNA levels of SHP were significantly increased (all P<0.01), while LRH-1 and CYP7a1 were not changed in the ZR4.00G, ZR1.00G and ZR0.25G groups. Compared with the negative transfection group, the protein and mRNA levels of CYP7a1 and LRH-1 were significantly suppressed in the OE group (all P<0.01). Compared with the OE group, the protein and mRNA levels of SHP were evidently increased in the OE4G and OE1G groups (all P<0.01), while LRH-1 and CYP7a1 were not changed in the OE4G, OE1G and OE0.25G groups.
 Conclusion: The over-expression of LRH-1 in RPH can up-regulate the mRNA and protein levels of CYP7a1. GPA can improve the biochemical and liver pathology of ANIT-induced cholestasis rats, which may be related to the decrease of CYP7a1 by activating SHP through LRH-1 in RPH.
Animals ; Cholestasis ; Iridoid Glucosides ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; Signal Transduction

Objective To screen an ssDNA aptamer for rabbit mesenchymal stem cells (MSCs) and to identify the ability of the aptamer to recognize MSCs of a variety of species origin.Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively.Aptamers were screened by cell SELEX (systematic evolution of ligands by exponential enrichment) technique targeting to isolated MSCs.Enrichment of the 5th pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool was then cloned into pGE-T vector and the cloned sequences were determined randomly.The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit, rat, and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions.Aptamer 5-1-12 from 5th enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit, rat and human origin.Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.

Objective To observe the damage effect of borneol on rabbit corneal epithelial cells. Methods After the treatment with borneol at 100, 200, 400 μg·mL-1 respectively, the viability of rabbit corneal epithelial cells was determined by methyl thiazolyl tetrazolium ( MTT) assay, cell apoptosis was determined by flow cytometry with Annexin V- fluorescein isothiocyanate/propidium iodide ( FITC/PI) staining, and Caspase-3 mRNA expression was detected with real-time reverse transcription polymerase chain reaction ( RT-PCR) . Results Borneol at the concentrations of 100, 200, 400 μg·mL-1 inhibited the activity of rabbit corneal epithelial cells. Compared with the normal control group, borneol increased the rate of apoptosis, and enhanced the Caspase-3 mRNA expression in rabbit corneal epithelial cells ( P<0.05 or P<0.01) . Conclusion Borneol can inhibit the proliferation of rabbit corneal epithelial cells, induce cell apoptosis through enhancing the expression of apoptosis-related gene Caspase-3 mRNA.

OBJECTIVETo screen of high cariogenicity Streptococcus mutans (S. mutans) strains isolated from clinical specimens preliminary.

METHODSAcidogenicity, aciduricity, extracellular polysaccharide production and adhesion of 41 strains of S. mutans isolated from clinical specimens were investigated to screen high cariogenicity S. mutans strains.

RESULTSThere were different cariogenicity among 41 strains of S. mutans, in which 3 strains of S. mutans had all high ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid, indicated there were 3 strains with high cariogenicity S. mutans strains isolated from clinical specimens. Another 3 strains of S. mutans with all low ability to produce extracellular polysaccharide, adhere to the saliva-coated hydroxyapatite, produce acid and tolerate acid indicated they were low cariogenicity S. mutans strains isolated from clinical specimens.

CONCLUSIONWe may have obtained high cariogenicity S. mutans strains isolated from clinical specimens.

Dental Caries ; Durapatite ; Humans ; Saliva ; Streptococcus mutans

OBJECTIVETo identify Streptococcus mutans (S. mutans) strains from clinical samples.

METHODSPlaque samples from caries-active and caries-free sites on enamel surfaces were obtained and cultivated for S. mutans isolation. Morphology, biochemistry, automatic microorganism analysis system and polymerase chain reaction using primers homologous to surface protein antigen I/II (spaP), glucosyltransferase B (gtfB) and dextranase (dexA) were used to identify S. mutans. Genotype of isolated S. mutans was determined by arbitrarily primed polymerase chain reaction.

RESULTSForty-six strains of S. mutans were obtained from the 32 subjects and were identified as S. mutans by biochemistry, automatic microorganism analysis system and polymerase chain reaction. Five identical genotypes were found by arbitrarily primed polymerase chain reaction.

CONCLUSIONForty-one strains of S. mutans with different genotype were obtained from clinical samples.

Dental Caries ; Dental Plaque ; Genotype ; Glucosyltransferases ; Humans ; Polymerase Chain Reaction ; Streptococcus mutans

OBJECTIVETo select and identify ssDNA aptamers specific to Streptococcus mutans strains with different cariogenicity isolated from clinical specimens.

METHODSSubtractive SELEX technology targeting the whole intact cells was used to screen for ssDNA aptamers specific to the clinical isolates Streptococcus mutans strains with different cariogenicity. Radioactive isotope, flow cytometry, gene cloning and sequencing, MEME online software and RNA structure analysis software were employed to analyze the first and secondary structures of the aptamers and identify the screened aptamers.

RESULTSDetection by radioactive isotope showed sufficient pool enrichment after 9 rounds of subtractive SELEX. Flow cytometry showed that the selected aptamers H1, H16, H4, L1, L10 and H19 were capable of binding specifically with highly cariogenic Streptococcus mutans strains but not with strains with a low cariogenicity. The aptamer H19 had the strongest binding capacity to highly cariogenic Streptococcus mutans strains, with a dissociation constant of 69.45∓38.53 nmol/L.

CONCLUSIONWe have obtained the ssDNA aptamers specific to the clinical isolates of highly cariogenic Streptococcus mutans strains.

Aptamers, Nucleotide ; genetics ; Cloning, Molecular ; DNA Primers ; Dental Caries ; microbiology ; Gene Library ; Humans ; Nucleic Acid Conformation ; SELEX Aptamer Technique ; Species Specificity ; Streptococcus mutans ; classification ; genetics ; isolation & purification

Objective To explore the optimal method of extraction and determination of rhynchophylline and isorhynchophylline in Ramulus Uncariae cum Uncis. Methods A RP-HPLC was performed on a Phenomenex C18 column (4. 6 mm× 150 mm, 5m) at 30℃. The mobile phase consisted of methanol-0. 01 mmol/L triethylamine solution (70: 30, pH 7.0 adjusted by glacial acetic acid) at the flow rate of 0. 5 mL/min. The automatic sample injector was set at 5℃ and the ultraviolet detector was operated at 245 nm. And then, the effect of different extraction condition on the contents of rhynchophylline and isorhyn-ehophylline in Ramulus Uncariae cum Uncis was investigated. ResultsA good linearity of rhynchophylline was in the range of 2. 5μg/mL to 80. 0μg/mL (Y=76. 7170X-0. 0727,r=0. 999 8),the average recoveries were from 99. 84 ％ to 116. 91％, and the RSD (n=6) were less than 8.8 ％. A good linearity of isorhynchophylline was in the range of 2. 0 μg/mL to 80. 0 μg/mL (Y=87. 4729X-0. 3666, r=0.999 7), the average recoveries were from 87.08 ％ to 104. 97 ％, and the RSD (n=6) were less than 7.3 ％. The contents of rhynchophylline and isorhynchophylline in the methanol-extracted solu- tion were approximately ten times as much as those in water-decocted solution. The main factors which had effect on the ex-traction of rhynchophyUine and isorhynchophylline in methanol solution were supersonic time,methanol amount and cold-dousing time, in a decreasing sequence. The best condition selected by orthogonal experiment wasb as follows: extracting the medicinal material with 20-fold volumes of methanol, cold maceration for 24h and supersonic extraction for 1 h. Conclusion The extraction percentage of rhynchophylline and isorhynchophylline in the extraction condition of cold maceration with methanol and supersonic extraction is superior to that in the water-decocting condition. The method is simple, fast and accurate, and it can be used for the quality control of Ramulus Uncariae cum Uncis.

Human cytomegalovirus glycoprotein complex Ⅱ (gC Ⅱ ) consists of two glycoproteins, gM and gN. Although gC Ⅱ specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report on the generation of virus-neutralizing antibodies by immunizing with one epitope of gM. The epitope, termed MAD, was screened from random phage peptide library by subtractive strategy. The peptide sequence of MAD was highly homologous with 32~38 amino acids of HCMV gM. Mice immunized with MAD coupled with keyhole limpet hemocyanin (KLH) could produce specific antibodies against MAD, and the antibodies obtained could bind not only native HCMV particles, but also the recombinant gM30～78 peptide. ELISA analysis results showed that MAD could specifically bind HCMV-positive human serum samples. Virus-neutralizing assay results demonstrated that the antibodies against MAD could inhibit HCMV strain AD169 entering the human embryonic lung cells. The results suggested that MAD could be used as a new potential protective antigen in the development of HCMV vaccine.

We identified the critical amino-acid residues in antigen M derterminant (MAD) epitope of human cytomegalovirus protein M. On the basis of the peptide sequence of MAD, some conservative residues were mutated into the glycine residue. Then the gene fragment of mutants linked to amino terminal of Fc were cloned into the plasmid pET32-Fc and expressed by fusion with Fc. After purified by protein A affinity chromatography, the activity of mutants binding the goat polyclonal antibodies against human cytomegalovirus (HCMV) were detected by ELISA and Western blotting. Our results showed that when glutamine residue was mutated into glycine residue, the activity of MAD(Q --> G) binding the goat polyclonal antibodies against HCMV was reduced apparently. Other mutants did not have the same characteristics. The activity of MAD was closely related to the conformation of glutamine residue.
Amino Acids ; genetics ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Base Sequence ; Cytomegalovirus ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Goats ; Humans ; Immunoglobulin Fc Fragments ; genetics ; immunology ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Mutation ; Viral Matrix Proteins ; genetics ; immunology

BACKGROUND: Borneol can open blood-brain barrier (BBB) but the mechanisms are not very clear. Histamine and 5-hydroxtryptamine can take part in regulation of permeability of BBB. There is not report on the rela tion between the effect of opening BBB of borneol and the regulation of permeability of BBB of histamine (HA)/5-hydroxytryptamine (5-HT). OBJECTIVE: To study the relation between the effect of opening BBB of bornool and the regulation of permeability of BBB of histamine (HA)/5-hydroxytryptamine (5-HT).DESIGN: A completely randomized and controlled study.SETTING: Clinical Pharmacological Institute of University of Traditional Chinese Medicine of Guangzhou.MATERIALS: The experiment was carried out at the Clinical Pharmacological Institute of University of Traditional Chinese Medicine of Guangzhou from September 2003 to February 2004. Totally 104 healthy male SD rats, weighting 230-27.0 g, supplied by Guangdong Medical Experimental Animal Center, were selected.METHODS: The rats were randomly divided into 13 groups: pre-medicine group and post-medicine group (high, moderate and low dosage group and 5, 20, 45 and 60 minutes group in each dosage group) with 8 in each group. Borneol was mixed as 10% millet oil suspension. Rats were fasted before experiment for whole night, and medicine was perfused on the next morning with the high, moderate and low dosage of 0.15, 0.12 and 0.09 g/kg respectively. Hypothalami of rats was selected at various time points to make biological samples. Contents of HA and 5-HT were assayed with HPLC system electrochemical detector.MAIN OUTCOME MEASURES: Changes of level of histamin (HA) and 5-HT in hypothalamus of rats after administration of borneol.RESULTS: Data of totally 104 rats was entered the final analysis. ① Contents of HA and 5-HT in hypothalami were 2.07±0.54 μg/g and 1.45±0.14 μg/g respectively. ② The level of HA in hypothalamus of rats after different doses of borneol were higher than that of before administration. Comparing the level of HA in 20 minutes after moderate dose with before administration, the level of HA in 20 minutes was increased significantly [(3.36±0.21) μg/g, P < 0.01], the others of moderate dose, the 45 minutes after high dose, the 20 and 45 minutes after low dose were also increased significantly than before administration (P < 0.05). ③ After administration of different doses of borneol, the level of 5-HT after high dose were higher than that of before administration [5, 20, 45 and 60 minutes after medicine: (1.90±0.32), (3.28 ±0.25), (2.66±0.46), (2.80±0.34) μg/g, respectively; (P < 0.05 or P < 0.01)]; the level of 5-HT after 5, 20 and 45 minutes of moderate and low dose were increased significantly too (P < 0.05 or P < 0.01).CONCLUSION: Borneol could open BBB by increasing levels of HA and 5-HT in hypothalamus of rats.Borneol mediates opening of BBB by increasing levels of HA and5-HT in rats.