The genetic analysis of herpesviruses has been a constant challenge, due to the large, complex genomes of herpesviruses and mutagenesis of viral genes by conventional recombination methods in cell culture. Recently, a completely new approach for full-length infectious clones of herpesviruses based on bacterial artificial chromosomes (BACs) has been developed. This technique allows the maintenance, propagation and genetic modification of the viral genome as a BAC plasmid in E.coli, thus making the procedures fast, safe and effective in prokaryotic cells. This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells, thereby facilitating the analysis of viral gene functions in the context of genome. In this presentation, Epstein-Barr virus was used as an example to describe the principle, establishment of the technique and mutation introduction into the BAC plasmid, and to discuss the perspective in the use of BAC-cloned herpesviruses.

This study aims to investigate the relationship between odor and contents of the chemical compounds in Lonicera japonica, including chlorogenic acid, galuteolin and polyphenols. High performance liquid chromatography (HPLC) was applied to determine the contents of chlorogenic acid and galuteolin in L. japonica. The ponptent of polyphenols was determined by UV-Vis Spectrophotometry. Electronic nose was used to extract and measure the odor of L. japonica. Then SPSS 17.0 software was employed for data processing. There is a significant positive correlation between the comprehensive index value of aroma and the contents of chlorogenic acid and polyphenols. The regression equations have been established. However, the relationship between the comprehensive index value and the content of galuteolin is not obvious. This is proof that the odor of L. japonica has close connection with the chemical compounds. Therefore, this research offered a new method for initially determine or predict the content of the chemical composition in L. japonica,
Chlorogenic Acid ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Electronic Nose ; Lonicera ; chemistry ; Odorants ; analysis ; Polyphenols ; chemistry ; Smell

OBJECTIVETo compare the clinical efficacy differences between acupoint catgut embedding and Kuntai capsule for perimenopausal syndrome, so as to provide an effective treatment method for perimenopausal syndrome.

METHODSThirty-three cases in the embedding group were treated with acupoint catgut embedding at back-shu points and front-mu points of liver, spleen and kidney combined with syndrome differentiation and disease differentiation, ten days per times; the Kuntai group was treated with oral administration of Kuntai capsule, 4 capsules each time, three times per day. The Kupperman index (KI) was observed in the two groups before treatment after 10 days, 30 days and 60 days of treatment, respectively; the efficacy was evaluated according to the ratio of KI.

RESULTSAfter the treatment, as treatment proceeded, the score of KI and ratio of KI were gradually reduced in two groups; the score of KI and ratio of KI in the embedding group after 10 days of treatment was lower than those in the Kuntai group (both P<0.05); after 10 days of treatment, the total effective rate was 36.4% (12/33) in the embedding group, which was superior to 3.0% (1/33) in the Kuntai group (P<0.05); however, after 30 days and 60 days of treatment, the differences of each index between two groups were not statistically significant (all P>0.05).

CONCLUSIONBoth the acupoint catgut embedding and Kuntai capsule could reduce the score of KI and improve clinical symptoms, and the acupoint catgut embedding has certain advantage on the early stage of treatment.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; Adult ; Catgut ; Female ; Humans ; Middle Aged ; Perimenopause ; physiology ; Treatment Outcome

The paper aimed to study the residue decline dynamic and standards for safety utilization of carbendazim in roots, stems, leaves of Anoectochilus roxburghii and in growth media. Samples extracted with methanol were purified by liquid-liquid extraction and analysed by HPLC. The results showed that average rate of recovery was 82.9% - 95.7% and RSD were 2.0% - 6.3% with add of carbendazim in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of carbendazim were treated, varying from recommended dosage (1.0 kg x hm(-2)) to 1.5 times recommended dosage (1.5 kg x hm(-2)). Results of two years test showed that the half-life period of carbendazim were 7.01 - 8.51 d in the growth media of A. roxburghii, 3.58 - 4.27 d in stems and 3.50 - 3.91 d in leaves, 4.93 - 5.71 d in roots. Providing max recommended residue of carbendazim in the cultivation of A. roxburghii is 0.5 mg x kg(-1), sprayed 4 times a year with the dosage of 1.0 kg x hm(-2), 28 days is proposed for the safety interval of the last pesticide application's and harvest's date.
Benzimidazoles ; metabolism ; pharmacology ; Carbamates ; metabolism ; pharmacology ; Chromatography, High Pressure Liquid ; Culture Media, Conditioned ; chemistry ; Dose-Response Relationship, Drug ; Fungicides, Industrial ; metabolism ; pharmacology ; Liquid-Liquid Extraction ; Orchidaceae ; drug effects ; metabolism ; Pesticide Residues ; analysis ; metabolism ; Plant Leaves ; drug effects ; metabolism ; Plant Roots ; drug effects ; metabolism ; Plant Stems ; drug effects ; metabolism

Background Congenital fibrosis of extraocular muscles (CFEOM) affects patient' s appearance and quality of life,and no effective treatment for this disease is available.Imaging study is helpful for exploring the pathogenesis of CFEOM.Objective This study was to describe the characteristics of CFEOM associated with limb movement disorder using magnetic resonance imaging (MRI).Methods A family with CFEOM associated with limb movement disorder was investigated in Renmin Hospital of Wuhan University.Disease history was collected and the pedigree was investigated.Ophthalmologic examinations,including corrected visual acuity,refractive error,slitlamp examination,ophthalmoscopic examination,force of levator palpebrae superioris,ocular movement,eye position,forced duction test,and bell phenomenon examination,were performed.Ocular orbital and cranial MRI was performed in 4 CFEOM patients and 10 normal subjects to compare the structures of the extraocular muscles,motor nerve and cranium.Oral informed consent was obtained from each patient prior to any medical examination.Results A total of 1 1 members from 3 generations were investigated in this study,presenting with 4 cases of disease.The mode of inheritance of this family complied with the Mendelian autosomal dominant inheritance law.Clinical signs included disturbance of eye movement,deviation of eye position,ptosis,lack of Bell sign and positive reaction of passive pull test.In addition,unstable gait,improper body limb alignment,dysphasia and mental retardation were ohserved in 1 patient,which coincided with the diagnostic criteria of type 3 CFEOM.MRI results demonstrated that the levator palpebrae superioris,superior rectus and superior oblique muscle were clearly thinner,and the medial rectus,lateral rectus,inferior rectus muscle were thinning in the patients,showing significant differences in comparison with the normal controls(P＜O.05).The oculomotor and abducens nerves became thinner and even absent in the patients.Cranial MRI showed that Ⅲ-3 in the pedigree with callosum was shorter than that of the normal controls,suggesting that patient suffered from corpus callosum hypoplasia.Meanwhile,cranial MRI indicated the presence of cerebellar hypoplasia and the expansion of the fourth ventricle.Conclusions MRI demonstrates consistent abnormalities of the oculomotor nerves and abducens nerves in the affected individuals in this CFEOM 3 family,and some affected members exhibit two types of central nervous system abnormalities-corpus callosum and cerebellar hypoplasia.These findings suggest that CFEOM 3 is primarily a neuronal disease.

Objective To analyze obese children serum lipid level in order to understand the relationship between serum lipid and cardiovascular disease in obese children.Methods One hundred and fifty-three children(109 male and 44 female)aged 4-16 years old with obesity who attended the outpatient clinic of Beijing Children′s Hospital were collected.Percentage body fat (%BF),body fat (BF),fat-free mass (FFM) was estimated by using bioelectrical impedance analysis (BIA) and calculate.Waist and hip circumference,waist-to-hip ratio (WHR) was estimated by soft tape measure and calculate.Skinfold thickness of scapular bone below (S) and triceps muscle (T),S/T rate was estimated by skin fold meter and calculate.Serum total cholesterol (TC),triglyceride(TG),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),apolipoprotein(Apo) AI and Apo B levels were also measured.SPSS 11.0 software was used to analyzed the data.Results The cardiovascular disease related was the prevalence of high TC levels(3.3%)or high LDL-C level(6.0%) and high TG level(24.7%) was rather low.HDL-C level was reduced in 31.3% of obese children.In children over 10 years old,%BW and %BF showed a weak correlation with HDL-C(r=-0.202,-0.211).Conclusions In obese children,serum lipid as well as Apo level should be exa-mined in order to evaluate angiocardiopathy.

ObjectiveTo investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning.Methods Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2×107 TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA).Results Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value):0.237±0.075 vs. 0.674±0.011, PON1 protein (gray value): 0.602±0.086 vs. 0.998±0.124, Nrf2 mRNA (gray value): 0.089±0.012 vs. 0.126±0.010, GSH (mg/g): 3.84±0.33 vs. 5.52±0.40, SOD (μg/g): 0.383±0.040 vs. 0.564±0.052, CAT (ng/g): 7.32±1.28 vs. 12.46±1.54, allP< 0.05], and remarkable increase in MDA content (nmol/g: 7.78±0.41 vs. 2.34±0.25,P< 0.05). With the extension of time, PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels gradually increased, MDA content gradually decreased, Nrf2 mRNA expression level had risen to the level of control group at 24 hours (0.133±0.019 vs. 0.126±0.009,P> 0.05), and it was higher than that of the control group at 48 hours (0.206±0.028 vs. 0.124±0.010,P< 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726±0.021 vs. 0.237±0.075, PON1 protein (gray value): 0.739±0.050 vs. 0.602±0.086, Nrf2 mRNA (gray value): 0.158±0.007 vs. 0.089±0.012, GSH (mg/g): 4.30±0.22 vs. 3.84±0.33, SOD (μg/g): 0.454±0.062 vs. 0.383±0.040, CAT (ng/g): 8.98±1.02 vs. 7.32±1.28, allP< 0.05], and a decrease in MDA content (nmol/g: 6.56±0.44 vs. 7.78±0.41,P< 0.05).Conclusion Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.

AIMTo study the effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts.

METHODSCultured L-6TG cells were divided into 3 groups: control group (C), ischemia/reperfusion group (I/R), PMA + ischemia/ reperfusion group (PMA), SOD, XOD and free calcium and mitochondrial respiration in L-6TG cell were evaluated in each group. Apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis, the immunohistochemical method was used to determine the expression of caspase-3.

RESULTSCompared with I/R group, in PMA group, XOD , free calcium in L-6TG cell and apoptotic percentage all decreased significantly, while SOD and mitochondrial respiration in L-6TG cell increased. DNA fragmentation analysis of L-6TG cell showed no laddering pattern. The expression of caspase-3 was down regulated significantly.

CONCLUSIONActivation of PKC can lessen ischemia/reperfusion injury and apoptosis through lessening oxidative injury and mitochondrial injury, adjusting calcium dyshomeostasis and down expression of caspase-3.

Animals ; Apoptosis ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cells, Cultured ; Mitochondria ; metabolism ; Myoblasts, Skeletal ; cytology ; metabolism ; Oxidative Stress ; Protein Kinase C ; metabolism ; Rats ; Reperfusion Injury ; metabolism ; pathology

AIMTo establish a model of ischemia/reperfusion injury on L-6TG cell.

METHODSCultured L-6TG cells were divided into 2 groups: control group (C), ischemia/reperfusion group (I/R), LDH in culture fluid, SOD, XOD, free calcium in L-6TG cell and mitochondria respiration were evaluated in each group, the micromorphologic changes were observed with microscope.

RESULTSCompared with control group, after L-6TG cell suffered ischemia 4 hours and reperfusion 4 hours, LDH in culture fluid, XOD, free calcium in L-6TG cell all increased significantly, while SOD in L-6TG cell and mitochondrial respiration decreased, structural damage to L-6TG cell was severe.

CONCLUSIONUsing mimicking ischemic solution and mimicking reperfusion solution can successfully establish a model of ischemia/reperfusion injury on L-6TG cell.

Animals ; Cells, Cultured ; Colorimetry ; L-Lactate Dehydrogenase ; analysis ; Muscle, Skeletal ; blood supply ; Rats ; Reperfusion Injury

OBJECTIVETo investigate the effects of the Yifuning soft gelatin capsules(YFN)on estrogen receptor (ER) and nitric oxide (NO) levels in ovariectomized rats.

METHODFifty female mature sprague-dawley rats were randomly divided into 5 groups: normal control; model control; diethylstilbestrol tablets (DT); YFN (high dose and low dose). After 4 weeks' treatment, the serum E2 levels were detected by radioimmunoassay. The estrogen receptor (ER) levels of uterus and artery were detected with method of radioligand binding assay of receptor (RBAR). The uterus pathologic changes were investigated with light microscope. NO and NOS levels of the artery and the uterus index were detected too.

RESULTYFN can obviously improve serum E2, increase index and ER of uterus (P < 0.01 or P < 0.05), and ameliorate the uterus' pathologic changes in OVX rats. It also can increase the artery' ER, NO and NOS levels.

CONCLUSIONYFN can cure the climacteric syndrome and prevent the cardiovascular disease after post-menopause.

Animals ; Aorta ; metabolism ; Capsules ; Curcuma ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Estradiol ; blood ; Female ; Materia Medica ; administration & dosage ; isolation & purification ; pharmacology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Ovariectomy ; Oviducts ; chemistry ; Plants, Medicinal ; chemistry ; Rana temporaria ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; metabolism ; Uterus ; metabolism ; pathology