Research Progress on Lichongtang in Treatment of Uterine Leiomyoma / 中国实验方剂学杂志

Uterine leiomyoma （UL）， the most common benign tumor of the reproductive system in women of childbearing age， is characterized by clinical symptoms such as increased menstrual flow， prolonged menstrual period， breast tenderness，backache， lower abdominal pain and mass in the lower abdomen. With the continuous progress of modern society， the age of women's marriage and childbirth is gradually pushed back， which to a certain extent has led to an increase in the probability of modern women suffering from UL. Relevant literature shows that the incidence of UL is about 70%， and 25%-50% of the patients have clinical symptoms， seriously endangering women's physical health. The prevention and treatment of UL by modern medicine is currently limited to two aspects： drug control of estrogen and progesterone levels and surgical removal. Traditional Chinese medicine（TCM）has shown obvious advantages in improving the clinical symptoms of UL patients， with very broad application prospects as it can regulate body's Qi and blood on the basis of syndrome differentiation， treatment and overall concepts. Lichongtang， as a famous TCM prescription for replenishing Qi， activating blood and removing blood stasis， was created by ZHANG Xi-chun， a famous Chinese medicine doctor in the Qing dynasty， and recorded in the Records of Tradition Chinese and Western Medicine in Combination. It is widely used in the field of gynecological diseases in clinical practice. Studies have shown that Lichongtang is effective in treating UL. Clinical observations show that Lichongtang can significantly relieve the clinical symptoms of UL patients such as prolonged menstrual period， dysmenorrhea， waist and abdomen swelling and irregular vaginal bleeding， with the characteristics of stable curative effect， high safety， less side effect and low recurrence rate. The experimental results show that Lichongtang has a comprehensive regulatory effect on UL through inhibiting the proliferation of UL cells and inducing apoptosis， reducing serum estrogen and progesterone level， regulating the apoptosis pathway of tumor cells， and promoting the degradation of extracellular matrix（ECM）. After retrieval in PubMed， CNKI and other databases， the authors made a review by summarizing the theories， clinical efficacy and action mechanisms of Lichongtang in the treatment of UL， in order to provide reference for the follow-up in-depth study of pharmacological mechanism of Lichongtang and its further clinical application and promotion.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer,and study its value on early diagnosis and prognosis evaluation.Methods Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital,Hubei University of Medicine were selected.Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay.The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve.The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model.Kaplan-Meier method was used for drawing the survival curve,and log-rank test method was used for comparison.Results The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group:(85.52 ± 12.18) ng/L vs.(66.20 ± 8.28) ng/L,and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group:(76.44 ± 7.56) ng/L vs.(66.20 ± 8.28) ng/L,and there were statistical differences (P ＜ 0.05).ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L,and area under curve (AUC) was 0.869,with a sensitivity of 73.75％ and a specificity of 91.67％;the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L,and AUC was 0.827,with a sensitivity of 78.57％,and a specificity of 78.33％.According to the median serum ULBP2 level,the patients were divided into ULBP2 high expression (ULBP2 ＞ 85.52 ng/L,38 cases) and ULBP2 low expression (42 cases).The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P ＜ 0.05).Univariate Cox regression analysis result showed that lymph node metastasis,distant metastasis,tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P ＜ 0.01 or ＜ 0.05);multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR =0.194,95％ CI 0.077 to 0.490,P =0.001).The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs.50 months),and there was statistical difference (P ＜ 0.05).Conclusions Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective@#To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer, and study its value on early diagnosis and prognosis evaluation.@*Methods@#Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital, Hubei University of Medicine were selected. Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay. The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve. The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model. Kaplan-Meier method was used for drawing the survival curve, and log-rank test method was used for comparison.@*Results@#The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group: (85.52 ± 12.18) ng/L vs. (66.20 ± 8.28) ng/L, and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group: (76.44 ± 7.56) ng/L vs. (66.20 ± 8.28) ng/L, and there were statistical differences (P<0.05). ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L, and area under curve (AUC) was 0.869, with a sensitivity of 73.75% and a specificity of 91.67%; the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L, and AUC was 0.827, with a sensitivity of 78.57%, and a specificity of 78.33%. According to the median serum ULBP2 level, the patients were divided into ULBP2 high expression (ULBP2>85.52 ng/L, 38 cases) and ULBP2 low expression (42 cases). The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P < 0.05). Univariate Cox regression analysis result showed that lymph node metastasis, distant metastasis, tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P<0.01 or <0.05); multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR = 0.194, 95% CI 0.077 to 0.490, P = 0.001). The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs. 50 months), and there was statistical difference (P<0.05).@*Conclusions@#Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Expresión génica de ligandos mica, micb y ulbp (1-6) del receptor NKG2D de células natural killer y metaloproteinasas adam10, adam17 y mmp14 en lineas celulares de cáncer de cervical / Gene expression of mica, micb and ulbp (1-6) ligands of the NKG2D receptor of natural killer cells and metalloproteinases adam10, adam17 and mmp14 in cells lines of cervical cancer

RESUMEN El CCU es la segunda causa de muerte en mujeres de nuestro país. Dentro de los primeros mecanismos de defensa del hospedero se encuentra la respuesta inmune de las células NK y su función lítica a expensas de su receptor activador NKG2D, el cual posee como ligandos mica, micb y ulbp (1-6), los cuales se expresan en células transformadas y/o infectadas por virus. Uno de los mecanismos de evasión por parte de la célula tumoral es el clivaje de estas proteínas a través de metaloproteinasas como adam10, adam17 y mmp14. Se analizó la expresión de estos ligandos y metaloproteinasas mediante PCR tiempo real, en lineas celulares de referencia para cáncer cervical como HeLa (positiva para VPH-18) y C33A (negativa para VPH). Se obtuvieron valores representativos de expresion relativa genica con diferencias significativas asi: mmp14 en linea HeLa (p= 0.006); y mica y ulbp-3 en la linea C33A (p= 0.020 y p=0.003 respectivamente). Por lo tanto, se podría sugerir que la expresión de mmp14 se encuentra posiblemente involucrados con la presencia de VPH causante del cancer cervical y la respuesta inmunne innata desarrollada.

ABSTRACT Cervical cancer is the second leading cause of death in women in our country. Within the first host defense mechanisms is the immune response of NK cells and their lytic function at the expense of its NKG2D receptor activator which has as ligands mica, micb and ulbp (1-6), which are expressed in transformed cells and / or virally infected. One of the mechanisms of evasion by the tumor cell is the cleavage of these proteins through metalloproteinases as adam10, adam17 and mmp14. We analyzed the expression of these ligands and metalloproteinases by real time PCR, in reference to cell lines HeLa cervical cancer (positive for HPV-18) and C33A (negative for HPV). We obtained representing relative gene expression with significant differences from the other lines of study as follows: mmp14 in HeLa (p = 0.006); and mica and ulbp-3 in C33A (p = 0.020 and p = 0.003 respectively). Thus one might suggest that the expression of mmp14 is possible involved with HPV presence causing high risk of cervical cancer and innate inmunne response developed.

Evaluation of Tolerable Upper Intake Levels for Vitamin D in Children and Adolescents

In 2012, the European Food Safety Authority (EFSA) suggested a tolerable upper intake level (UL) for vitamin D at 100 µg/day for adults based on the risk of hypercalcaemia. EFSA concluded that consumption of up to 50 µg/day does not lead to hypercalcaemia in children and adolescents (10-17 years). Furthermore, EFSA stated that there is no reason to assume that children and adolescents in the phase of rapid bone formation and growth have a lower tolerance for vitamin D compared to adults, and a UL of 100 µg/day for adolescents aged 11-17 years and 50 µg/day in children 1-10 years, taking the smaller body size into account, was proposed. The Norwegian Food Safety Authority (NFSA) is currently revising the national regulation of maximum limits in food supplements (not yet harmonised in the European Economic Area (EEA)), including maximum limits for vitamin D. NFSA has therefore requested the Norwegian Scientific Committee for Food Safety (VKM) to evaluate the assumption in the EFSA opinion that children and adolescents can tolerate the same amount of vitamin D as adults due to rapid bone formation and growth. In children and adolescents with lower weight than adults, this assumption actually implies that adolescents can tolerate more vitamin D per kg body weight than adults. VKM is therefore requested to evaluate if there is scientific evidence that a UL at 50 µg/day for children (1-10 years) and 100 µg/day for adolescents (11-17 years) is safe. The present statement is prepared by members of the Panel on Nutrition, Dietetic Products and Novel Food and Allergy in VKM. Three literature searches were performed to find new relevant studies investigating high intakes of vitamin D in children and adolescents and the role of vitamin D in bone formation and growth. No studies supporting a higher tolerance to vitamin D in children and adolescents due to rapid bone formation and growth were retrieved in the literature search. Moreover, there is apparently no firm association between bone formation and vitamin D levels in children during their growth period into adolescence and adulthood. No studies investigating high intakes of vitamin D in children 1-10 years were found. Furthermore, no studies that have examined safety issues and/or adverse effects of vitamin D supplementation in doses above 50 µg/day in adolescents were identified. It can therefore not be concluded that the UL at 50 µg/day in children (1-10 years) and 100 µg/day in adolescents (11-17 years) is safe. In the 2002 report from European Scientific Committee on Food (SCF), a UL was set at 25 µg/day for children aged 2-10 years, and 50 µg/day for adolescents aged 11-17 years (corresponding to the UL for adults at that time). To the best of knowledge no serious, harmful effects have been reported for these doses of vitamin D.

An overview of Crimean-Congo hemorrhagic fever in Pakistan

Crimean-Congo Hemorrhagic Fever (CCHF) is a disease affecting domestic livestock and wild animals which can spread to humans. It is caused by infection with a tick- borne virus (Nairovirus) in the family Bunyaviridae or by contacting with infected tissues or from animal blood. CCHF cases were recorded from published data from 2013 to 2018 in different geographic regions of Pakistan. The intensity and risk factors were also determined from all four provinces of Pakistan. A total of 391 cases of CCHF have been reported from all over Pakistan during period of 2013-2018. Majority of them were recorded at the time of Eid-ul-Adha. CCHF cases were identified predominantly in Baluchistan (n=12), Karachi (n=5), Bahawalpur (n=2), and Khyber Pakhtunkhwa (n=1). The prevalence of disease were different in different areas of Pakistan (Fata 1%, Islamabad 5%, Punjab 21%, Sindh 8%, KPK 14% and Baluchistan 39%). The political disturbances faced by the Pakistan have increased Pakistan's susceptibility because large number of refugees have migrated to Pakistan from Afghanistan which is an endemic country. Most of the immigrants and their cattles from Afghanistan settled in Khyber, Pakhtunkhwa and Baluchistan provinces which ultimately cause higher prevalence of CCHF in these arears. Currently there is no complete cure or commercially available vaccine of CCHF available in Pakistan. Mostly Ribavirin antiviral drug is used to treat CCHF. The disease can be controlled by implementing preventive measures like avoiding contact with blood of the suspected animal and tick bites.

Glaucoma due to ciliary body cysts and pseudoplateau iris: a systematic review of the literature / Glaucoma associado a cistos de corpo ciliar e íris pseudoplateau: revisão sistemática da literatura

ABSTRACT The diagnosis of angle-closure glaucoma secondary to iridociliary cysts is challenging and lacks compiled literature support. We present a rare case of bilateral angle-closure glaucoma associated with pseudoplateau iris due to multiple ciliary cysts and conducted a systematic review of the literature to find similar case reports published between November 2006 and November 2016. Only 19 case reports present treatment modalities, and most cases required more than one therapeutic approach for controlling the intraocular pressure. Pseudoplateau iris attributed to iridociliary cysts should be considered in the differential diagnosis of patients with narrow angles, particularly those with ocular hypertension and glaucoma, in which management is complex. In addition to gonioscopy, ultrasound biomicroscopy is considered the conclusive method for accurate diagnosis.

RESUMO O diagnóstico de glaucoma de ângulo fechado secundário a cistos iridociliares é desafiador e não possui suporte da literatura compilada. Apresentamos um caso bilateral raro de glaucoma de ângulo fechado associado à íris pseudoplateau devido a cistos ciliares múltiplos e realizamos uma revisão sistemática da literatura de relatos de casos similares publicados entre novembro de 2006 e novembro de 2016. Apenas 19 relatos de casos apresentaram as modalidades de tratamento e na maioria deles foi necessário mais de uma abordagem terapêutica para controlar a pressão intra-ocular. Íris pseudoplateau atribuída a cistos iridociliários deve ser considerada no diagnóstico diferencial de pacientes com ângulos estreitos, particularmente aqueles com hipertensão ocular e glaucoma, em que o manejo é complexo. Além da go nioscopia, a biomicroscopia ultra-sônica é considerada o método conclusivo para o diagnóstico correto.

Anti HSV-1 effect of camptothecin by inhibiting HSV-1 replication genes / 中草药

Objective To study the antiviral effect and mechanism of camptothecin against HSV-1 in vitro. Methods Adding different concentration of camptothecin to the Vero cells infected by HSV-1, the measure of cytopathologic effects (CPE) was taken to evaluate the result of antiviral effect and mechanism of camptothecin. The median toxic concentration (TC50), median inhibitory concentration (IC50), and therapeutic index (TI) of camptothecin were calculated, and the affection of camptothecin on HSV-1 mRNA expression was examined through the method of RT-qPCR. Results TC50 of camptothecin was 2 153.44 nmol/L, IC50 was 43.01 nmol/L, and TI was 50.07. The results of RT-qPCR showed that camptothecin can significantly inhibit the gene transcription of HSV-1, UL12, UL42, UL54, and TK, and its inhibitory effect was in dose-dependence relationship with the concentration of camptothecin. Conclusion Camptothecin has an obvious antiviral effect on HSV-1 and can inhibit the transcription of HSV-1 genes such as UL12, UL42, UL54, and TK.

Vacuolar H+-ATPase regulates vaginal microenvironment / 中国医师杂志

Objective The purpose of this study was to develop the mechanism of vacuolar H+-ATPase regulation of vaginal microenvironment.Methods In this research,53 women were divided into three groups.Their age,serum estradiol (E2),serum follicle stimulating hormone (FSH),vaginal pH value,and mRNA expression of vacuolar H +-ATPase (VHA) on the ectocervical-vaginal epithelial cells were analyzed.Results (1) As serum E2 levels decreased,the vaginal pH values increased and VHA mRNA declined (P ＜ 0.01).(2) Immunohistochemistry scores decreased in the three groups.VHA expression decreased in human ectocervical-vaginal epithelial tissues except basal cells.(3) The expression of VHA was positive correlated with estradiol,while negative correlated with age and vaginal pH value (P ＜ 0.01).Conclusions Estradiol could regulate the genetic transcription and synthetic of VHA protein under vaginal microenvironment.

Effect of Shuanglong formula on no-reflow in rats with myocardial ischemia/reperfusion / 中国药理学通报

Aim To explore the effect of Shuanglong formula(SLF) on no-reflow in rats with myocardial is-chemia/reperfusion (I/R). Methods The rats were divided into five groups, namely, sham group, I/R group,SLF(5,2.5,1.25 g·kg-1)group. Treatment group received SLF decoction by gavage once a day for five days,while other groups were offered drinking wa-ter by gavage once a day for five days. The rats in I/R group and SLF-pretreated group were induced by iga-tion of left anterior descending coronary artery,and the rats were subjected to ischemia for 4h followed by reperfusion. Sham operation group did not undergo oc-clusion of the coronary artery. After 4 hours' reperfu-sion, real-time myocardial contrast echocardiography was used to monitor regional blood perfusion and cardi-ac functions. Blood was collected from the abdominal aorta and the serum was separated, and the levels of cTnT, CRP, CK and LDH were measured. The myo-cardial no-reflow area and infarction area were assessed by thioflavin S and nitrotetrazolium blue chloride, re-spectively. Results The SLF-pretreated group exhibi-ted significant reductions in the infarct area and no-re-flow area compared with I/R group(P <0.01 or P <0.05). In SLF-pretreated groups, β, A and A·β significantly increased as compared to those in I/R group. The LV anterior wall systolic and diastolic thicknesses (LVAW d/s) were significantly improved in SLF-pretreated group compared with those in I/R group. The LV internal diameter in systole (LVID s) and the LV volume in systole(LV s) were significantly reduced in SLF-pretreated group compared with those in I/R group. The EF, FS and SV were significantly improved in SLF-pretreated group compared with those in I/R group. The comparison between SLF-pretreated group and I/R group showed no significant difference in LDH, CK, cTnT, and CRP levels. Conclusion Shuanglong formula minimizes the sizes of myocardial infarct area and no-reflow area,improving regional my-ocardial blood flow and cardiac function.

In vitro expression of human cytomegalovirus UL148 RNA and prediction of its functional sites / 中华微生物学和免疫学杂志

Objective To express UL148 RNA of human cytomegalovirus (HCMV) clinical strains in vitro and to study its functions. Methods Urine of a newborn with HCMV infection was inocula-ted into human embryo lung cells. HCMV clinical strain was isolated and identified by multiplex PCR. UL148 gene was amplified and cloned into pGEM-T-Easy plasmid after double enzyme digestion. A recombi-nant plasmid was constructed and located at the downstream of the T7 promoter. The recombinant plasmid was identified by electrophoresis of the recombinant plasmid,PCR product and double enzyme product. Se-quencing analysis was used for final confirmation. UL148 was transcribed into RNA by 32P labeling. Post-translational modification sites were analyzed by bioinformatics method based on UL148 sequence characteris-tics. Results The clinical strain of HCMV was obtained in vitro. Electrophoresis and sequencing analysis confirmed the successful construction of the recombinant plasmid. UL148 RNA was transcribed in vitro by T7RNA polymerase. Post-translational modification sites showed that UL148 gene contained one cell adhe-sion sequence, one legume lectins beta-chain signature, two N-myristoylation sites, one casein kinase Ⅱphosphorylation site,seven protein kinase C phosphorylation sitse, one cAMP/cGMP-dependent protein ki-nase phosphorylation site, two N-glycosylation sites and one transmembrane region. Conclusion UL148 gene might encode a viral adhesion molecule involving in the signal transduction in host cells.

Screening and Analysis of Proteins Interacting with HCMV UL145 from a Human Fetal Brain cDNA Library / 中国医科大学学报

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Characterization of the sequences of human cytomegalovirus UL141 in low-passage clinical isolates in Guan-gzhou / 医学研究生学报

Objective The sequence characteristics and polymorphism of the UL141 gene may help find the pathogenesis of human cytomegalovirus (HCMV) infection.This study was to charac-terize the sequences of HCMV UL141 in low-passage clinical isolates in Guangzhou . Methods We collected urine samples from 10 in-fants with clinically confirmed HCMV infection in the Guangzhou are-a, isolated low-passage clinical virus strains and identified them by multiplex PCR.We performed amplification, cloning, identification and sequencing of the HCMV UL141 gene and searched the GenBank for its homologous sequences followed by sequence analysis . Results The HCMV UL141 gene was found to have 2 open reading frames( ORF) , UL141a and UL141b, composed of 315 and 1017 nucleotides and included in the GenBank with the sequence numbers of DQ180372 and DQ180371, respectively.The full length of the UL141 gene in the low-passage HCMV clinical strain (D3) was 1277 bp, the coding protein consisting of 338 amino acid residues , and the full lengths of the ORFs UL 141a and UL141b were 315 bp and 1017 bp, respectively, with relatively conservative DNA sequences .Mutations were identified in 46 sites with base substitution but no insertion or deletion .The modification sites in all the isolated strains were relatively conservative after translation of the HCMV UL 141 coding protein.The isoelectric points of the UL141 protein were 8.36-8.68 for all the clinical isolates. Conclusion Polymorphism exists in the UL141 gene and its amino acid sequences of the HCMV low-passage clinical strains isolated from infants in Guangzhou , which has shed some light on the function of the ULl 41 protein and pathogenesis of HCMV infection .

Features of human cytomegalovirus UL146 gene in asymptomatic children / 中华微生物学和免疫学杂志

Objective To investigate the polymorphisms of human cytomegalovirus ( HCMV ) UL146 gene in asymptomatic children. Methods Urine samples were collected from 47 asymptomatic chil-dren who were positive for HCMV DNA. PCR was performed to amplify the open reading frame ( ORF) of UL146 gene. Positive bands were sequenced and variations in UL146 gene were analyzed by using bioinfor-matics software. Results Seventeen samples were successfully amplified and sequenced. Variations spread all over the sequence of UL146 gene and the variability in nucleotide and amino acid sequences ranged from 0％ to 42. 5％ and 0％ to 67. 7％ respectively. Compared with the Towne strain, there was diversity in sig-nal sequence and C-terminal region. Phylogenetic analysis indicated that UL146 in the 17 asymptomatic chil-dren belonged to four genotypes, which were G1, G8, G9 and G11. Forms of post-translational modification varied greatly among the four genotypes, while the important functional region of ELRCXC chemokine was highly conservative. Secondary structure prediction showed that random-coli conformation was the predomi-nant structure of active proteins. Isoelectric point ( PI) and molecular weight ( MW) were dissimilar among the four genotypes. Conclusion HCMV UL146 gene in asymptomatic children was hypervariable in both nucleotide sequence and amino acid structure. However, the important functional region was highly con-served. The predominant genotypes of UL146 in these children were G1, G8, G9 and G11, and the geno-type distribution in them showed no significant difference with previous findings in children with symptomatic HCMV infection.

Characterization of the sequences of human cytomegalovirus UL141 in low-passage clinical isolates in Guan-gzhou / 医学研究生学报

Objective The sequence characteristics and polymorphism of the UL141 gene may help find the pathogenesis of human cytomegalovirus (HCMV) infection.This study was to charac-terize the sequences of HCMV UL141 in low-passage clinical isolates in Guangzhou . Methods We collected urine samples from 10 in-fants with clinically confirmed HCMV infection in the Guangzhou are-a, isolated low-passage clinical virus strains and identified them by multiplex PCR.We performed amplification, cloning, identification and sequencing of the HCMV UL141 gene and searched the GenBank for its homologous sequences followed by sequence analysis . Results The HCMV UL141 gene was found to have 2 open reading frames( ORF) , UL141a and UL141b, composed of 315 and 1017 nucleotides and included in the GenBank with the sequence numbers of DQ180372 and DQ180371, respectively.The full length of the UL141 gene in the low-passage HCMV clinical strain (D3) was 1277 bp, the coding protein consisting of 338 amino acid residues , and the full lengths of the ORFs UL 141a and UL141b were 315 bp and 1017 bp, respectively, with relatively conservative DNA sequences .Mutations were identified in 46 sites with base substitution but no insertion or deletion .The modification sites in all the isolated strains were relatively conservative after translation of the HCMV UL 141 coding protein.The isoelectric points of the UL141 protein were 8.36-8.68 for all the clinical isolates. Conclusion Polymorphism exists in the UL141 gene and its amino acid sequences of the HCMV low-passage clinical strains isolated from infants in Guangzhou , which has shed some light on the function of the ULl 41 protein and pathogenesis of HCMV infection .

Features of human cytomegalovirus UL146 gene in asymptomatic children / 中华微生物学和免疫学杂志

Objective To investigate the polymorphisms of human cytomegalovirus ( HCMV ) UL146 gene in asymptomatic children. Methods Urine samples were collected from 47 asymptomatic chil-dren who were positive for HCMV DNA. PCR was performed to amplify the open reading frame ( ORF) of UL146 gene. Positive bands were sequenced and variations in UL146 gene were analyzed by using bioinfor-matics software. Results Seventeen samples were successfully amplified and sequenced. Variations spread all over the sequence of UL146 gene and the variability in nucleotide and amino acid sequences ranged from 0％ to 42. 5％ and 0％ to 67. 7％ respectively. Compared with the Towne strain, there was diversity in sig-nal sequence and C-terminal region. Phylogenetic analysis indicated that UL146 in the 17 asymptomatic chil-dren belonged to four genotypes, which were G1, G8, G9 and G11. Forms of post-translational modification varied greatly among the four genotypes, while the important functional region of ELRCXC chemokine was highly conservative. Secondary structure prediction showed that random-coli conformation was the predomi-nant structure of active proteins. Isoelectric point ( PI) and molecular weight ( MW) were dissimilar among the four genotypes. Conclusion HCMV UL146 gene in asymptomatic children was hypervariable in both nucleotide sequence and amino acid structure. However, the important functional region was highly con-served. The predominant genotypes of UL146 in these children were G1, G8, G9 and G11, and the geno-type distribution in them showed no significant difference with previous findings in children with symptomatic HCMV infection.

Production and Its Anti-hyperglycemic Effects of γ-Aminobutyric Acid from the Wild Yeast Strain Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1

This study was done to produce γ-aminobutyric acid (GABA) from wild yeast as well as investigate its anti-hyperglycemic effects. Among ten GABA-producing yeast strains, Pichia silvicola UL6-1 and Sporobolomyces carnicolor 402-JB-1 produced high GABA concentration of 134.4 µg/mL and 179.2 µg/mL, respectively. P. silvicola UL6-1 showed a maximum GABA yield of 136.5 µg/mL and 200.8 µg/mL from S. carnicolor 402-JB-1 when they were cultured for 30 hr at 30℃ in yeast extract-peptone-dextrose medium. The cell-free extract from P. silvicola UL6-1 and S. carnicolor 402-JB-1 showed very high anti-hyperglycemic α-glucosidase inhibitory activity of 72.3% and 69.9%, respectively. Additionally, their cell-free extract-containing GABA showed the anti-hyperglycemic effect in streptozotocin-induced diabetic Sprague-Dawley rats.

Screening and identification of proteins interacting with HCMV UL132 protein / 吉林大学学报(医学版)

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

Expression and polyclonal antibody preparation of the tegument protein UL48 encoded by MDV / 中国兽医学报

UL48 plays essential role in replication of MDV genome and interacts with UL36 as well as other MDV tegument proteins.To investigate the interaction between UL48 and UL36 during MDV oncogenisis,antibody against UL48 was prepared and characterized in current study.UL48 gene was amplified from MDV-Ⅰ genome and then subcloned into pTYB1 and pGEX-4T3 vectors for UL48 expression with induction of IPTG in BL21(DE3) E..coli cells.Chitin-sepharose and Glutathion-sepharose were,respectively,used to purify fusion protein intein-UL48 and GST-UL48.Four subcutaneous injections of intein-UL48 fusion protein were done on the lower back and the thigh of rabbit and then other three injections with an interval 10 days.The titer of antibody was measured by the sandwich ELISA with UL48 protein isolated from GST-UL48 after cleavage of thrombin.Western blot was carried out for specificity analysis of antibody against UL48 protein.The results suggested that UL48 antibody was succesfully prepared,and its titer was 1 ∶ 512 000.

Effective inhibition of human cytomegalovirus UL148 gene expression by external guide sequences in vitro / 中华实验和临床病毒学杂志

Objective@#To investigate the UL148 gene function of human cytomegalovirus (HCMV) low passage clinic isolate and new strategies for anti-HCMV treatment, the DNA-based external guide sequences (EGSs) were designed to inhibit UL148 RNA expression.@*Methods@#UL148 RNA secondary structure was analyzed by RNA structure technique, an appropriate region was chosen for DNA-based EGS57 synthesis, targeted the UL148 RNA. The M1RNA and UL148 RNA were generated by PCR for transcription in vitro. The UL148 RNA and M1RNA were transcribed in vitro under the function of T7 RNA polymerase. The UL148 was labelled by 32P. The cleavage reactions were carried out by mixing up EGS, M1RNA with UL148 RNA for 1 h. The products were separated by urea denaturing polyacrylamide gel electrophoresis and detected with Typhoon Phosphor Imager.@*Results@#UL148 RNA ranged from 58 to 72 sites was the binding position, and 57 was a cleavage site. EGS57 was designed and synthesized. EGS57 was combined with UL148 RNA to form the natural substrate of M1RNA. UL148 RNA and M1RNA were synthesized through T7 RNA polymerase catalyzing, and the products were conformed. After cleaving reactions, DNA-based EGS57 was shown to be able to cleave UL148 RNA efficiently in vitro by a complex with M1RNA.@*Conclusions@#UL148 RNA was cleaved efficiently by EGS57, and the cleaving site was conformed as expectation. It will provide the gene silent tool effectively for further study the function of UL148 gene.