Recombination plasmid pMM085 possessed both immunogens heat-labile enterotoxin(LT) and fimbriae antigen K88 of enterotoxigenic Escherichia coli (ETEC). Althouth vaccine strain MM-3 carrying pMM085 had good effect to protect piglets against diarrhea due to ETEC infections,it was not ideal live vaccine for pMM085 bringing chloramphenicol resistance gene (cat). To solve the problem,the host-plasmid balanced lethal system was introduced which including the replacement of cat gene by asd gene and transformation the new plasmid to the strain X6097 which asd gene was knocked out in its chromosome. Considering pMM085 was a big plasmid (23kb) and traditional genetic manipulations was not easy to carry on,?-Red recombination system was adopt in this work to realize the replacement of cat gene by asd gene. The results indicated that ?-Red recombination system was convenient and efficient to reconstruct big plasmid.

OBJECTIVE: To investigate characteristics of molecular etiology of children with profound sensorineural hearing loss in Hubei province, and to provide reference for deafness treatment and genetic counseling. METHOD: Three hundred and six children with profound sensorineural hearing loss in Hubei province were enrolled, their genomic DNA were extracted from peripheral blood and a deafness gene test chip was used to screen nine hot spot mutation in the GJB2, GJB3, SLC26A4, and mitochondria 12SrRNA gene. All patients with SLC26A4 gene mutation were given temporal bone CT scan. RESULT: One hundred and thirty-two (43.14%) out of 306 children were found carrying at least one pathogenic gene mutation. The mutation rates of GJB2, SLC26A4 and mitochondria DNA 12SrRNA gene were 29.41% (90/306), 13.72% (42/306) and 0.65% (2/306), respectively. None out of 306 children was detected GJB3 gene mutation. Thirty-six patients carrying SLC26A4 gene mutation were detected enlarged vestibular aqueduct by CT scan. CONCLUSION Mutations of GJB2 and SLC26A4 gene are two major pathogenic gene for genetic hearing loss in children. 235delC mutation is the main mutation type, followed by IVS7-2A> G mutation type. The screening of SLC26A4 gene common mutations contribute to the diagnosis of enlarged vestibular aqueduct syndrome.
Adolescent ; Child ; Child, Preschool ; China ; Connexin 26 ; Connexins ; genetics ; DNA Mutational Analysis ; Deafness ; genetics ; Female ; Genetic Testing ; Humans ; Infant ; Male ; Membrane Transport Proteins ; genetics ; Mutation ; Oligonucleotide Array Sequence Analysis ; Sulfate Transporters

OBJECTIVETo analyze and compare molecular mechanisms of active ingredients of honeysuckle (chlorogenic acid, CGA) with bovine lactoferrin (BLF) or bovine serum albumin (BSA).

METHODThe spectral experiment and the computer analog technology were combined to determine the binding parameters, energy transfer parameters and thermodynamic functions between CGA and proteins, study the molecular mechanism, and compare the differences in interactive mechanism between CGA and BLF or BSA.

RESULTThe interactive mechanism between CGA and BLF or BSA was a dynamic molecular mechanism, whereas the static quenching mechanism existed between the interaction of CGA and BSA, with differences in the bonding intensity due to difference temperature. The binding distance r between CGA and BLF/BSA was very short, indicating the phenomenon of energy transfer. The results of the molecular modeling showed that the main interaction force between CGA and BLF or BSA was hydrogen bonds, together with Van der Waals' forces and hydrophobic effect.

CONCLUSIONThe computer analog shows consistent results with spectral experiment.

Absorption ; Animals ; Cattle ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Humans ; Hydrophobic and Hydrophilic Interactions ; Lactoferrin ; chemistry ; metabolism ; Lonicera ; chemistry ; Models, Molecular ; Protein Binding ; Protein Conformation ; Serum Albumin, Bovine ; chemistry ; metabolism ; Thermodynamics

OBJECTIVETo study the effect of qingxin kaiqiao formula and saponin on the learning and memory ability and the expression of the apoptosis signal transducers Abeta and betaAPP in AD rat brain.

METHODThe comparative observation method was adopted for the animal test. Forty male SD rats were randomly divided into five groups, namely the normal group, the model group, the aricept group, the qingxin kaiqiao formula group and the saponin group, with eight rats in each group. Abeta(25-35) (10 g x L(-1)) was injected into their bilateral amygdala to establish the AD rat model. Since the next day, they were intragastrically administered with Aricept (1.67 mg x kg(-1)), Qingxin Kaiqiao decoction (12.67 mL x kg(-1)), saponin (6.30 mg x kg(-1)) and double distilled water filling for 2 weeks to observe their spatial memory ability in a Morris water maze and study the expression of Caspase-3, Abeta and betaAPP in brain tissues by immunohistochemistry.

RESULTEach traditional Chinese medicine groups showed significant improvement in the learning and memory ability of AD rats and notable differences (P < 0.05, P < 0.01) compared with the control group. The qingxin kaiqiao formula group and the saponin group showed a decrease in the expressions of Caspase-3, Abeta and betaAPP in cerebral cortex and hippocampus area, displaying notable differences (P < 0.01, P < 0.05) compared with the control group.

CONCLUSIONqingxin kaiqiao formula and saponin can obviously improve the learning and memory ability of AD rats with by decreasing the expression of Caspase-3, Abeta and betaAPP in cortex and hippocampus.

Alzheimer Disease ; drug therapy ; genetics ; Amyloid beta-Peptides ; genetics ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cerebral Cortex ; drug effects ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Learning ; drug effects ; Male ; Maze Learning ; drug effects ; Memory ; drug effects ; Rats ; Rats, Sprague-Dawley ; Saponins ; administration & dosage ; pharmacology ; Time Factors

OBJECTIVETo show the space structures of Mingmen (GV 4) area and its adjacent tissues.

METHODSA frozen female corpse was cut into about 0.5 mm thick sections with a high-speed steel plane machine and photographed with digital and optical camera. Two 2-D transverse phase picture data sets were obtained and then were sequenced, matched, modified and divided. The 3-D reconstruction software was used to reconstruct the outline form of the lumbar part containing Mingmen (GV 4) to attain transverse phase picture data sets (3 022 sheets, 0.57 mm in thickness). The sagittal, coronal and different oblique plane were cut and the tissue structures in these pictures were analyzed. Thus, the reconstruction data sets were obtained.

CONCLUSIONAfter the structures of Mingmen (GV 4) were reconstructed with computer, the form of Mingmen (GV 4) can be dynamically observed from any angle and the space construction law of gross form of Mingmen (GV 4) can be proved.

Lumbosacral Region ; Software ; Tomography, X-Ray Computed

Objective: To observe the clinical efficacy of Zhen'ai needling method in Nei Jing (Classic of Internal Medicine) for children with allergic rhinitis (AR) accompanied by adenoid hypertrophy (AH). Methods: A total of 74 children who met the screening criteria were divided into a Zhen'ai group and a control group by the random number table method, with 37 cases in each group. The control group was treated with acupuncture at Zusanli (ST 36), Hegu (LI 4), Yingxiang (LI 20), Juliao (ST 3), Yintang (GV 29), Shangxing (GV 23) and Baihui (GV 20). The Zhen'ai group added points of Zhen'ai needling method {Shanglianquan [Extra, located at 1 cun above Lianquan (CV 23)], Tianrong (SI 17) and Lieque (LU 7)} in addition to the points in the control group. The needles were retained for 30 min. The treatment was performed twice a week. The total nasal symptom score (TNSS), sino-nasal outcome test-20 (SNOT-20) and symptom scale for AH (SSAH) were assessed before and after 10 treatments. The clinical efficacy of the two groups was compared after treatment. Results: During the treatment, 5 cases dropped out in the control group and 2 cases in the Zhen'ai group. After treatment, the total effective rate of the Zhen'ai group was 94.3％, versus 93.8％ in the control group. There was no significant difference between the two groups (P>0.05). The markedly effective rate of the Zhen'ai group was 42.9％, versus 12.5％ in the control group, and the difference was statistically significant (P<0.05). After treatment, there were significant intra-group differences in the scores of TNSS, SNOT-20 and SSAH in both groups (all P<0.05); the scores of SNOT-20 and SSAH in the Zhen'ai group were lower than those in the control group, and the differences between the groups were statistically significant (both P<0.05). Conclusion: Both conventional acupuncture and conventional acupuncture plus Zhen'ai needling method can improve clinical symptoms of children with AR accompanied by AH; and conventional acupuncture plus Zhen'ai needling method has a better effect than conventional acupuncture in improving AH symptoms.

AIM: To study the chemical constituents of the roots of Ilex asprella Champ. ex Benth. METHODS: Compounds were isolated by silica gel, ODS, and Sephadex LH-20 column chromatography, and their structures were elucidated on the basis of physicochemical properties and spectroscopic analysis. RESULTS: Four triterpenoid glycosides were isolated and identified as (3β)-19-hydroxy-28-oxours-12-en-3-yl β-D-glucopyranosiduronic acid n-butyl ester (1), ilexasoside A (2), monepaloside F (3), and ilexoside A (4). CONCLUSION Compound 1 is a new triterpenoid glycoside, and compounds 3 and 4 were isolated from this plant for the first time.
Glycosides ; chemistry ; isolation & purification ; Ilex ; chemistry ; Molecular Structure ; Plant Extracts ; chemistry ; isolation & purification ; Plant Roots ; chemistry ; Triterpenes ; chemistry ; isolation & purification

Sorafenib is a multi -targeted tyrosinekinase inhibitor , and mainly used for the treatment of advanced renal cell carcinoma and hepa -tocellular carcinoma.The pharmacokinetic changes of sorafenib caused by drug -drug interactions may affect its clinical efficacy and safety, so individualized treatment drug monitoring could control the occurrence of potential drug interactions.In this paper, the clinical applications and drug -drug interaction of sorafenib was reviewed .

OBJECTIVENeonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) which resulted from mutation in SLC25A13 gene can present transient intrahepatic cholestasis, low birth weight, growth retardation, hypoproteinemia and so on. This study aimed to identify the mutation type of NICCD patients by DNA sequencing.

METHODSTwenty children diagnosed as NICCD were consented to enroll in this study. PCR assays were performed to amplify the eighteen exons and its flanking sequences of SLC25A13 gene, which were defined as the upstream and downstream 50 bp from starting and ending site of the exons. Then the PCR products were purified and followed by automated DNA sequencing. The IVS16ins3kb mutation was detected by nested PCR and RT-PCR.

RESULTSSeven genetic variations of SLC25A13, termed as 851del4, 1638ins23, IVS16ins3kb, IVS6+5G>A, c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C), were identified in the subjects, of which c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C) were reported for the first time in NICCD patients. And a compound mutation of［1638ins23+IVS16ins3kb］was also identified. In 20 patients with NICCD, 6 patients were 851del4 homozygotes, 7 patients were compound heterozygotes, and 7 patients were heterozygotes of single mutation. 851del4 was the major mutation type (64%), followed by 1638ins23 (15%), IVS16ins3kb (12%) and IVS6+5G>A (6%).

CONCLUSIONS851del4 is the major mutation type in Chinese patients with NICCD.

Cholestasis, Intrahepatic ; genetics ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Mitochondrial Membrane Transport Proteins ; deficiency ; genetics ; Mutation ; Sequence Analysis, DNA

OBJECTIVETo assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

METHODSBased on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing.

RESULTSHRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing.

CONCLUSIONHRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.

Anion Transport Proteins ; genetics ; Base Sequence ; Calcium-Binding Proteins ; deficiency ; China ; Citrullinemia ; diagnosis ; genetics ; metabolism ; DNA ; chemistry ; genetics ; Genetic Predisposition to Disease ; Genotype ; Humans ; Mitochondrial Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Nucleic Acid Denaturation ; Organic Anion Transporters ; deficiency ; Sensitivity and Specificity