With homology cloning approaches coupling with RACE (rapid-amplification of cDNA ends) techniques, the full-length coding sequence of pathogenesis-related protein PR10-1 with differential expression was cloned from the total RNA of the root of Panax notoginseng, and its function was explored furtherly. As a result, the longest 465 bp ORF (named as PnPR10-1 with the Accession No. KJ741402 in GenBank) was detected from the cloned sequence with full-length of cDNA of 863 bp. The corresponding peptide encoded consisted of 155 amino acids, contained some domains such as Bet-v-I, and showed high similarity with that from Panax ginseng by analysis of phylogenetic trees created from the alignments. Real-time quantitative PCR showed that the expression of PnPR10-1 gene was constitutive in different tissues of 1-3 year old plant, suggesting that it might be involved in growth, development, and secondary metabolism; yet it was up-regulated significantly with the infection of Fusarium oxysporum in root, suggesting that it might be involved in defense against many diseases including root rot in P. notoginseng.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Genes, Plant ; Glycoside Hydrolases ; genetics ; Molecular Sequence Data ; Open Reading Frames ; Panax notoginseng ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Plant Roots

OBJECTIVETo investigate the expression changes and regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in corpus luteum during pregnancy.

METHODSPregnant rats' ovaries were collected at different time points. The techniques of RT-PCR and in situ hybridization were used to observe expression changes of PACAP mRNA in rat ovaries during pregnancy. To further explore the regulation mechanism of PACAP mRNA expression in corpus luteum, luteal cells were cultured in vitro. Immature (25 - 28 days old) female Sprague-Dawley rats were injected subcutaneously with 50IU pregnant mare serum gonadotrophin (PMSG), and 25IU human chorionic gonadotrophin (hCG) 48 h later, to induce follicular development and luteum formation. On day 6 after hCG administration (the day of hCG administration was the first day), the rats were killed by guillotine and the ovarian luteal cells were collected. After incubation for 24 h, luteal cells were administration with various factors for 24 h. And then expression changes of PACAP mRNA in luteal cells after administration with different factors were detected by RT-PCR, and radioimmunoassay was used to analyze progesterone levels.

RESULTSWith the development of pregnancy, the expression of PACAP mRNA increased gradually, reached the peak at pregnancy 19 d, and then decreased. Compared with control group, platelet activating factor (PAF), forskolin and PMA could obviously stimulate PACAP mRNA expression in luteal cells which were cultured with corresponding factors for 24 h. At the same time, progesterone levels in culture media were also elevated.

CONCLUSIONPACAP, acting as a local ovary regulator, was closely related to the maintenance of medium-term and late pregnancy. PAF could directly stimulate PACAP mRNA expression in luteal cells, and protein kinase C (PKC) and protein kinase A (PKA) signal pathways could both participate in this process.

Animals ; Cells, Cultured ; Corpus Luteum ; metabolism ; Female ; Pituitary Adenylate Cyclase-Activating Polypeptide ; genetics ; metabolism ; Platelet Activating Factor ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.
Cell Separation ; methods ; Culture Media ; Pinellia ; physiology ; Protoplasts ; physiology ; Regeneration

Based on a pair of specific primers, a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold I which carried the His tag, this recombinant plasmid was then determined by enzyme digestion, PCR and DNA sequencing. This recombinant plasmid pCold-Cap was transformed into E. coli Rosetta 2, and PGETV Cap fusion protein was expressed through IPTG induction. The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0. Immol/L IPTG under 15"C for 24h, the expression quantity was 40. 2%. The product had a molecular mass about 32. 3kD as expected. The target protein was separated in gel slices and used to immunize Balb/c mice. The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained. The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.
Alphavirus ; genetics ; immunology ; metabolism ; Alphavirus Infections ; immunology ; veterinary ; virology ; Animals ; Antibodies, Viral ; blood ; immunology ; Blotting, Western ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; DNA Primers ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Swine ; Swine Diseases ; immunology ; virology ; Zoonoses

OBJECTIVETo explore the effects of Fuzheng Paidu Tablet (FPT) on serum levels of tumor necrosis factor-alpha (TNF-alpha) and neopterin (NPI) in patients with asymptomatic human immunodeficiency virus (HIV) carriers.

METHODSUsing flow cytometry detection technology, CD T lymphocyte in anticoagulant blood sample of 32 asymptomatic HIV infection patients who were taking FPT for 6 months was detected and compared with before treatment. The serum levels of TNF-alpha and NPI were determined using ELISA method and compared with 22 healthy volunteers.

RESULTSAfter 6 months of treatment by FPT, the CD4+ T lymphocyte of asymptomatic HIV carriers increased from (368.63 +/- 111.54)/mm3 to (412.72 +/- 159.63)/mm3. Before treatment the serum levels of TNF-alpha [(20.05 +/- 13.08) nmol/L] and NPI [(9.55 +/- 2.52) nmol/L] were obviously higher than those of the healthy volunteers [(12.20 +/- 4.07) nmol/L and (5.91 +/- 1.43) nmol/L] (P < 0.05). After 6 months of treatment by FPT, they were lower after treatment than before treatment (P < 0.05). But there was no statistical difference in the serum TNF-alpha level [(11.06 +/- 4.71) nmol/L] when compared with the healthy volunteers group (P > 0.05). But the serum NPI level [(8.08 +/- 2.13) nmol/L] was still higher than that of the healthy volunteers group (P < 0.05).

CONCLUSIONSOne of the pathological factors for asymptomatic HIV infection is abnormal immune activation represented by increased serum levels of TNF-alpha and NPI. FPT could lower the serum levels of TNF-alpha and NPI in asymptomatic HIV infection patients, which was one of its possible mechanisms for its efficacy.

Adult ; Aged ; CD4 Lymphocyte Count ; Case-Control Studies ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; HIV Infections ; blood ; drug therapy ; Humans ; Male ; Middle Aged ; Neopterin ; blood ; Tablets ; Tumor Necrosis Factor-alpha ; blood

Objective To observe the changes in tear quantity and tear break-up time(BUT) before and after wearing overnight orthokeratology (OK),and investigate the influence of OK on tear film in young adolescents.Methods Prospective clinic study was conducted in 60 adolescents,aged 8-16 years,who met the inclusion criteria during November 2016 and May 2017 in the Second Hospital of Dalian Medical University were chosen and fitted with OK (OK group).At the same time,60 adolescents who met the same requirement were fitted with spectacle (spectacle group).The two groups were both divided into three subgroups according to the age (subgroup of 8-10 years old,11-13 years old,and 14-16 years old).The BUT,Schirmer' s I test score,and corneal fluorescent (FL) were measured and recorded before wearing and 1 week,1 month,3 months,6 months and 12 months after wearing.Results Totally 57 patients in the OK group (114 eyes) and 59 patients in the spectacle group (118 eyes) completed the follow-up study.No significant difference in Schirmer' s Ⅰ test scores was observed between the OK group and spectacle group (all P＞0.05).The BUT in OK group was significantly lower than that in the spectacle group at all the time points (all P ＜ 0.05).Of all subgroups,the subgroup aged from 8-10 years exhibited the sharpest and most rapid reduction in BUT.Conclusion Based on our findings,long-term OK wearing has no effect on tear volume but reduces the stability of tear film.The results suggest that OK should be considered to be a good alternative for children and adolescents with spectacle;however,it may influence the quality of tear film.In addition,special attention should be paid to younger adolescents,and regular follow-up is needed.

Objective To explore the heterosexual intercourse characteristics and its influencing factors among MSM population. Methods Voluntary counseling/testing and questionnaire survey were carried out at the bathhouses where MSM usually assemble. Results Among 342 MSM surveyed,52. 05% were married. 76 people( 22. 22%) were completely homosexual,while 266 people(77. 78%)were bisexual. 24(31. 57%)MSM who answered″ totally gay″ in self-assessment of sex orientation were married,significantly lower than those answered bisexual(57. 89%)(p<0. 05). 197 of participants had sex with women ,and 123 MSM had sex with women during the last six months,49 MSM of which used condoms at each heterosexual activity. MSM who were married or bisexual were easy to have heterosexual intercourse. Among the MSM surveyed,the HIV and syphilis infection rates were 11. 40% and 16. 67% respectively. There was no significant difference between the use of condoms and infection( both p>0. 05). In the recent six months,there was no significant difference in the use of condoms between infected( 43. 34%)and non-infected persons( 43. 34%)( p >0. 05). Conclusion Among MSM population,the prevalence of heterosexual intercourse is high,while condom use rate is relatively low. It is necessary to strengthen the behavioral intervention and promote condom use.

Objective:To explore an efficient method of practical teaching for ultrasonography.Methods:There were 41 five-year clinical medical students intending to learn ultrasonography in the spring semester of 2019. After completing common theory courses, they were divided into two groups randomly for practice courses, which consisted of two modules: Lecture and Hands-on. Lecture included case-based learning while Hands-on included pictures reading. Each group followed different sequence of modules. There were assessments of ultrasound theory, practice or the combination of both before, during and after practice courses.Results:The theory and practice ability of each group improved significantly after practice courses ( P < 0.05). Moreover, the group whose Hands-on module preceded Lecture module got the higher scores in the evaluation of the combined ability of theory and practice ( P < 0.05). Conclusion:The theory was as important as the practice during the clerkship of ultrasonography. The clinical practical teaching pattern that Hands-on precedes Lecture has achieved better teaching effects.

Acupuncturists have always paid high attention to deqi (needling sensation) in clinical. However, relationship between deqi and curative effect has not been elucidated yet. In order to have a further understanding on effect of deqi on clinical effect, through study on literature associated with deqi since the establishment of People's Republic of China in 1949, it is held that various factors can influence therapeutic effect of acupuncture treatment, including whether needling sensation appears or not, the speed of deqi, the intensity of deqi, different types of sensations as well as propagated sensation along meridians (PSM). Joint launched multidisciplinary researches should be carried out to reveal the influence principle of deqi on acupuncture effect, and to lay foundations for the further studies on mechanism of deqi.
Acupuncture ; Acupuncture Points ; Acupuncture Therapy ; Humans ; Meridians ; Qi ; Treatment Outcome

OBJECTIVETo investigate the clinical features, differential diagnosis and management of congenital stenosis of external auditory canal (CSEAC) with cholesteatoma.

METHODSThe clinical information for 10 cases of CSEAC with cholesteatoma was retrospectively reviewed.

RESULTSThe patients' ages ranged from 4.75 to 22 years (average 12 years). The diameter of the external auditory canal (EAC) was < 2 mm. All 10 ears had a history of postural fistulae or sinuses. Bone erosion of EAC was distinctly shown in high-resolution computed tomography (HRCT) of all cases, as well as soft tissue masses, which led to enlargement of the bony canals. All patients underwent canaloplasty; eight ears received hearing reconstructions at the same time. Cholesteatoma in EACs was confirmed during the operations, accompanied by compression and destruction of the post-superior and/or inferior bony wall. Postoperative pathologic examinations proved the diagnosis of cholesteatoma, and excluded any tissue of bronchial cleft cyst or fistula. After a follow-up 1 to 3 years, no recurrent cholesteatoma was found in any of the 10 cases. All reconstructed EACs were clean and smooth. The hearing levels in the eight ears that received hearing reconstructions improved 20 - 35 dBHL.

CONCLUSIONSIn CSEAC with cholesteatoma, the bony wall of EAC is most commonly involved. This involvement will lead to bone erosion of the EAC and may subsequently lead to the formation of postural or cervical sinuses. HRCT of temporal bone can show characteristic signs of soft tissue mass in EAC, with adjacent bone erosion.

Adolescent ; Child ; Child, Preschool ; Cholesteatoma ; complications ; diagnosis ; surgery ; Constriction, Pathologic ; congenital ; Cutaneous Fistula ; complications ; diagnosis ; surgery ; Ear Canal ; abnormalities ; Ear Diseases ; congenital ; diagnosis ; surgery ; Female ; Humans ; Male ; Retrospective Studies ; Young Adult