Humans ; Infant, Newborn ; Infant, Premature ; Infant, Premature, Diseases ; diagnosis ; microbiology ; prevention & control ; Mycoses ; diagnosis ; prevention & control

To establish induction and liquid culture system for hairy roots of Danshen (Salvia miltiorrhiza), Agrobacterium rhizogenes A4, LBA9402, 15834 as test bacterium were used to infect aseptic leaves of Danshen. The hairy roots were induced and positive transgenic hairy roots were selected with PCR using rolB and rolC as the target gene. Then hairy roots of S. miltiorrhiza were harvested and salvianolic acids were extracted with 70% methanol containing 1% formic acid. The content of salvianolic acid B (SalB) and rosmarinic acid (RA) were determined by HPLC. According to the above research results, the Danshen hairy roots induced by A. rhizogenes LBA9402 were inoculated into the following group of culture media: MSOH, MS, B5, and 6,7-V liquid media. Then the same methods of extraction and determination for the content of Danshen hairy roots were adopted. Last, the hairy roots of S. miltiorrhiza induced by A. rhizogenes LBA9402 were inoculated into the MSOH liquid media with different pH values. The content of salvianolic acid were extracted with 70% methanol containing 1% formic acid and determined by HPLC. As a result, three kinds of A. rhizogenes A4, LBA9402, 15834 could induce hairy roots and Ri plasmids were integrated into the genome of S. miltiorrhiza by PCR. Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid, which were (3.27 ± 0.37)% [including (1.04 ±0.36)% of RA and (2.22 ± 0.29)% of SalB] and (3.17 ± 0.20)% [including (0.92 ± 0.31)% of RA and (2.25 ± 0.26)% of SalB], respectively. Hairy roots induced by A. rhizogenes LBA9402 when they were cultured in MSOH liquid media produced much more salvianolic acid, which was (4.56 ± 0.36)%, including (1.12 ± 0.26)% of RA and (3.44 ± 0.23)% of SalB. Hairy roots induced by A. rhizogenes LBA9402 produced the most salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81, which was 4.85%, including 1.16% of RA and 3.69% of SalB. So Danshen hairy roots induced by A. rhizogenes LBA9402 and A4 produced much more salvianolic acid when they were cultured in MSOH liquid media with the pH value 4.81. The research had established the foundation on genetic engineering to improve the quality of S. miltiorrhiza.
Agrobacterium ; physiology ; Benzofurans ; analysis ; metabolism ; Cell Culture Techniques ; instrumentation ; methods ; Cinnamates ; analysis ; metabolism ; Culture Media ; chemistry ; metabolism ; Depsides ; analysis ; metabolism ; Drugs, Chinese Herbal ; analysis ; metabolism ; Plant Roots ; chemistry ; growth & development ; metabolism ; microbiology ; Salvia miltiorrhiza ; chemistry ; growth & development ; metabolism ; microbiology

Objective To study whether hyperthermia and ionizing radiation could produce combined effects on the neurobehaviors of neonatal rats.Methods These rats who were pregnant for 8 or 9 days received respectively total body irradiation by 60Co-rays .When the rats were pregnant for 10 days,the temperature of rat anus was kept at (37?0.5) ℃,(41?0.5) ℃,(42?0.5) ℃,respectively in warming boxes and lasted for 2,3,4,5 minutes.The neurobehaviors of neonatal rats were observed.Results In contrast to control group and 37 ℃group ,when pregnant rats were exposed to high temperature.the neonate rats were delayed for the appearance of four physiologic markers,the age of acquisition for four reflexes and the time of learning and memory(P

Objective In order to investigate the relationship between hearing loss and diabetic peripheral neuropathy( DPN ) via comparing the pure tone thresholds and vibration perception threshold(VPT) in type 2 diabetic patients and control person without diabetes. Methods 173 subjects including 138 type 2 diabetic patients(DM)and 35 non-diabetes controls were examined for VPT and hearing threshold. Nerve conducting velocity ( NCV )including sensory nerve conducting velocity( SCV )and motorial nerve conducting velocity( MCV )of diabetic patients were determined. The participants were divided into three groups: control group (n = 35 ), DM group without peripheral neuropathy( non-PN group, n = 74 ), and DM group complicated with peripheral neuropathy (PN group,n = 64 ). The clinical characteristics, biochemical parameters , the incidence of sensorineural hearing loss ( SNHL),pure tone threshold, and VPT were compared among three groups. At last, the relationship between hearing thresholds and NCV were analyzed. Results The incidence of hearing impairment of sensorineural type was 29.69% in PN group, which was significantly higher than that of non-PN group( 17.57% )and control group( 17. 14% ). There was significant differences in age, duration of diabetes, glycolated hemoglobin (HbA1c), glycolated serum albumin ( GA), Fasting blood glucose( FPG), 2h postprandial blood glucose( PPG), VPT, and hearing threshold among the three groups( all P＜0. 05 ). The value of hearing threshold increased significantly( all P＜0. 05 ) in 3 VPT subgroups with VPT≤ 15 V, VPT 16-25 V, and VPT ＞25 V. The Spearman correlation analysis showed median NCV was negatively correlated with hearing threshold on 1.00, 2.00, 4. 00, and 8. 00 kHz ( All P ＜ 0. 05 ). The logistic regression analysis indicated that the age( regression coefficient =0. 088, P＜0. 01 ) was the independent risk factor of SNHL, median nerve MCV ( regression coefficient = -0. 135, P = 0. 046 ) was the important influencing factor of SNHL. Conclusion Diabetic patients are more likely to suffer from impaired middle-frequency and high-frequency hearing, DPN in patients is often complicated with hearing impairment. Age and median nerve MCV were major risk factors of SNHL in diabetic patients.

Adenocarcinoma ; diagnosis ; pathology ; Carcinoma, Squamous Cell ; diagnosis ; pathology ; Cervical Intraepithelial Neoplasia ; diagnosis ; pathology ; Female ; Humans ; Precancerous Conditions ; diagnosis ; pathology ; Retrospective Studies ; Uterine Cervical Neoplasms ; diagnosis ; pathology ; Vaginal Smears ; methods

AIM: To comparatively analyze the application of laser confocal microscopy and corneal smear in the diagnosis of fungal keratitis.METHODS: Totally 77 patients (77 eyes) diagnosed as fungal keratitis were selected.Laser confocal microscopy and corneal smear examination were performed to observe the characteristics of the images, and the detection rate of fungus were compared between the two methods.RESULTS: Of the 77 patients, 66 eyes (86%) were positive and 11 eyes were negative detected by laser confocal microscopy;51 eyes (66%) were positive and 26 eyes were negative detected by corneal smear examination, the difference was statistically significant compared between two group (P<0.05).CONCLUSION: Laser corneal confocal microscopy is relatively safe and sensitive.If combined the application of laser corneal confocal microscopy and corneal smear examination in clinical, the diagnosis rate of fungal keratitis may be improved.

italic>Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic. Its main water-soluble components are rosmarinic acid (RA) and salvianolic acid B (SAB), which are produced by phenylpropanoid pathway. 4-Hydroxyphenylpyruvate reductase (HPPR) is a key enzyme in phenylpropanoid metabolism pathway. SmHPPR1 was cloned from S. miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1. On this basis, SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid (pHPL) was determined. SmHPPR1-overexpressing (SmHPPR1-OE) hairy roots of S. miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed. The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g^-1 dry weight. The concentration of RA, SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09, 1.29, 1.15 times of that in control-3, respectively, and the content of Danshensu was 36.26% of that in control-3. Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2. Protein-protein interaction indicated CYT (TR74706_c0_g1), NADP⁺ (TR26565_c0_g1) and NADP⁺ (TR68771_c0_g1) is the central node of the network and participated in metabolic process and cellular process. The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis, and SmHPPR1-overexpressing in hairy roots of S. miltiorrhiza could increase the concentration of salvianolic acids through synergistically regulating other pathway genes.

Background Leber hereditary optic neuropathy (LHON)is a common inherited eye disease,which generally affects young adults with bilateral loss of central vision.Mutation frequency of Leber hereditary has not been fully clarified. Objective This study was to investigate the mutation frequency of mitochondrial NDI gene associated with LHON in Chinese population. Methods The proposal of the study was approved by Ethic Committee of Wenzhou Medical College.Written informed consent was obtained from each subject initial of this trial.Eight hundred and ninety-four LHON patients and 134 normal subjects were collected.Genomic DNA was extracted from peripheral blood leukocytes of the all participants.Polymerase chain reaction (PCR) was used to amplify and sequence analysis of the mitochondrial ND1 gene was performed and aligned with revised Cambridge Reference Sequence(rCRS) of mitochondrial DNA.Then mutated gene frequency was screened and analyzed. Results Mutational analysis of mitochondrial ND1 gene in 894 LHON patients revealed the presence of G3316A,T3394C,G3460A,C3497T,G3635A,G3733A,and T4216C.11.19％ LHON patients (100/894 ) were found to be associated with the gene mutations mentioned above,and 3.24％ patients (29/894) showed the co-occurrence of three primary mutations.Mutation frequencies in LHON patients were 2.57％,2.23％,1.45％,3.80％,0.67％,0.11％,0.34％,respectively,and G3316A,T3394C,C3497T and T4216C also were detected in 134 normal controls with the mutation frequencies of 4.48％,2.99％,4.48％ and 1.49％,respectively.Mutation frequency analysis showed an insignificant difference in the mutations of G3316A,T3394C,C3497T and T4216C between LHON patients and normal controls (x2 =0.926,P=0.336;x2 =0.052,P=0.820; x2 =0.142,P=0.707;P=0.129).G3376A,G3496T,G3700A,A4136G,T4160C and C4171A were absent in Chinese LHON patients. Conclusions Mitoehondrial ND1 gene in LHON is a mutational hotspot in Chinese population,11.19％ (100/894)associated with LHON was caused by ND1 gene mutation.G3635A,G3733A may be rare pathological mutation in Chinese population.However,G3316A,T3394C,C3497T and T4216C are insufficient to produce the clinical phenotype,but they may play a synergic role for penetrance and phenotypic manifestation in LHON.

Objective To investigate the relationship between human leukocyte antigen-G( HLA-G) gene Exon 8 14 bp deletion polymorphism and the pathogenesis of severe pre-eclampsia.Methods Forty-two pregnant women with severe pre-eclampsia,who admitted to the Third Affiliated Hospital of Zhengzhou University from October 2008 to February 2009,and their newborns were chosen as the severe pre-eclampsia group.Another 45 healthy gravidas at the third trimester and their newborns were chosen as the control.All gravidas in both groups were Han Nationality.HLA-G Exon 8 genotyping was detected by PCR in both groups and the allele frequencies and genotype frequencies were compared between the two groups.The genotype frequencies of maternal-neonatal pairs were also analyzed.Results ( 1 ) In the severe pre-eclampsia group,14% of the maternal-neonatal pairs were homozygote of 14 bp deletion,and significantly higher frequency 33% (15/45) was found in the control group (P =0.038).(2) No significant difference was found in the allele frequencies and genotype frequencies of HLA-G 14 bp deletion polymorphism among all the mothers between the two groups (P >0.05).(3) The + 14 bp and-14 bp allele frequencies of HLA-G 14 bp deletion polymorphism in newborns in the severe pre-eclampsia group were 44% (37/84) and56% (47/84),respectively,and 30% (27/90) and 70% (63/90) in the control group.Although there was no significant difference between the two groups,but differences in trends was identified (χ2= 3.678 P = 0.055) ; The genotype (-14 bp/-14 bp) frequency of neonates in the severe pre-eclampsia group showed no difference compared with that in the control group[29% (12/42) vs 49% (22/45)],but differences in trends was also found (P =0.052).Conclusions HLA-G 14 bp deletion polymorphism is associated with the susceptibility of severe pre-eclampsia in Chinese Han nationality.Maternal-fetal genotype pairs of-14 bp/-14 bp may have reduced risk of severe pre-eclampsia.

OBJECTIVETo investigate the protective effect and action mechanism of petroleum ether extracts from Saussurea involucrate on brain tissues of hypoxia rats under constant pressure and closed conditions.

METHODThe PESI dosage-dependent experiment for hypoxia rats was conducted under constant pressure and closed conditions by intraperitoneally injecting 125, 250, 500 mg x kg(-1) to finalize that the optimum dosage is the high dose of PESI. Afterwards, 90 Wistar rats were randomly divided into the hypoxic model group, the acetazolamide 250 mg x kg(-1) group and the PESI high dose group. Each group was further divided into three subgroups according to different hypoxia times, with 10 rats in each subgroup. Under the same hypoxia and administration conditions, the rats were sacrificed after 0, 3, 6 h respectively. Their brain samples were collected for common pathological observation and immunohistochemical staining of HIF-1alpha. Real-time RT-PCR was used to detect HIF-1alpha, EPO, HO-1 and Caspase-3 gene expressions. And the Western blot assay was adopted to detect HIF-1alpha protein expression.

RESULTThe brain tissues of the hypoxia model group were severely damaged with the increase in the hypoxia time. The acetazolamide group and the PESI high does group were damaged in a much lower degree. According to the gene expression and the Western blot assay, high dose of PESI could inhibit HIF-1alpha expression. According to the pure gene expression test, high dose of PESI could increase EPO and HO-1 mRNA expressions, but inhibit Caspase-3 mRNA expression.

CONCLUSIONPESI's protective mechanism for brain tissues of hypoxia rats under constant pressure and closed conditions may be related to its effects in inhibiting HIF-1alpha expression, increasing EPO expression and resisting cell apoptosis.

Alkanes ; chemistry ; Animals ; Brain ; cytology ; drug effects ; metabolism ; Caspase 3 ; genetics ; Cell Hypoxia ; drug effects ; Cytoprotection ; drug effects ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Erythropoietin ; genetics ; Gene Expression Regulation, Enzymologic ; drug effects ; Heme Oxygenase-1 ; genetics ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Male ; Rats ; Rats, Wistar ; Saussurea ; chemistry