Clinical Effect of Tiaomai Mixture on Premature Ventricular Contraction in Coronary Heart Disease Due to Qi-Yin Deficiency and Stagnated Heat in Blood Vessel / 中国实验方剂学杂志

Objective:To investigate the clinical effect of Tiaomai mixture combined with metoprolol tartrate on premature ventricular contraction in coronary heart disease （CHD） due to Qi-Yin deficiency and stagnated heat in blood vessel. Method:A total of 95 patients with CHD complicated with premature ventricular contraction were randomized into a treatment group and a control group. Four cases dropped out， leaving 91 cases （45 in the treatment group and 46 in the control group） included in the follow-up. On the basis of routine treatments for CHD， patients in the control group were further treated with metoprolol tartrate， while those in the treatment group received metoprolol tartrate plus Tiaomai mixture. Such curative effect and safety indexes as traditional Chinese medicine （TCM） syndrome score， electrocardiogram （ECG）， and 24 h dynamic ECG were observed before and after four-week treatment. Result:After treatment， the therapeutic effect on arrhythmia in the treatment group was better than that in the control group（<italic>P</italic><0.05）. The treatment group was superior to the control group in reducing the frequency of premature ventricular contraction （<italic>P</italic><0.05）， improving the Lown grade （<italic>P</italic><0.01）， increasing the heart rate variability index （<italic>P</italic><0.05）， and ameliorating the QT dispersion in ECG （<italic>P</italic><0.05）， hypersensitive C-reactive protein， and homocysteine（<italic>P</italic><0.05）. As revealed by comparison with those before treatment， both interventions improved TCM syndrome， with better outcomes observed in the treatment group （<italic>P</italic><0.01）， manifested as the alleviation of shortness of breath， fatigue， dry mouth with desire to drink， and tongue and pulse manifestations （<italic>P</italic><0.01）. Conclusion:Tiaomai mixture improves the clinical efficacy against arrhythmia in CHD patients by regulating the heart rate variability index， inhibiting inflammatory cytokines， lowering homocysteine， and relieving clinical symptoms， which is worthy of clinical promotion and application.

Effect of Safety Bladder Capacity Catheterization on Lower Urinary Tract Function in Patients With Suprasacral Spinal Cord Injury / 中国康复理论与实践

Objective:To evaluate the effect of safety bladder capacity catheterization on lower urinary tract function in patients with supracacral spinal cord injury. Methods:A total of 60 patients with lower urinary tract dysfunction after suprasacral spinal cord injury in our hospital from January to December, 2019 were divided into control group (n = 30) and observation group (n = 30) randomly. Both groups were given intermittent catheterization, the frequency of catheterization was determined according to postvoid residual volume in the control group, while it was according to safety bladder capacity in the observation group. Their maximum destrusor pressure, postvoid residual volume, safety bladder capacity, urinary tract infection and detrusor wall thickness were compared. Results:Eight weeks after intervention, the maximum destrusor pressure and postvoid residual volume decreased, and the safety bladder capacity increased in the observation group (t > 5.623, P < 0.05), and were better than that of the control group (t > 2.242, P < 0.05); the detrusor wall thickness significantly decreased in the observation group (t = 7.871, P < 0.05), and was lower than that of the control group (t = 3.049, P < 0.01). The number of urinary tract infection patients was less in the observation group than in the control group (χ2 = 4.320, P = 0.038). Conclusion:Intermittent catheterization based on safety bladder capacity can improve lower urinary tract function in patients with suprasacral spinal cord injury.

Exploration and practice of micro-video teaching of / 中国针灸

In this paper, the micro-video teaching mode was explored in the course construction of . The micro-video teaching contents include the academic thought, experience in diagnosis and treatment, characteristic technology and clinical manipulation of famous acupuncture experts in the Henan University of CM. Each micro-video film is designed within 15-18 min, including three sections of knowledge, i.e. basic theory, technological application and clinical manipulation. Each section is designed within 5-6 min. The construction of the teaching course of is the innovation of practice mode of TCM and the new approach to the inheritance of the experience of experts. The construction of micro-video teaching course propels the reform of teaching mode, improves the learning initiative of students and clinical manipulative ability so as to improve the teaching effect and quality.

Progress of screening methods in vitro for HIV-1 capsid protein inhibitors / 中国药理学通报

Human immunodeficiency virus type 1 (HIV-1) infection is highly dependent on its conical fullerene capsid, which is composed of approximately 1 500 capsid proteins. In recent years, capsid protein has been recognized as an ideal target for design and screening drugs for AIDS therapy. Screening methods are the key to develop HIV-1 capsid inhibitors. A variety of screening approaches for HIV-1 capsid protein inhibitor, which have been reported in the literature are reviewed in the article.

Inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction / 中国中药杂志

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.

Serum Interleukin-6 Level and the rs1800795 Polymorphism in its Gene Associated with Neuroblastoma Risk in Chinese Children / 中华医学杂志(英文版)

<p><b>Background</b>The pro-inflammatory cytokine, interleukin-6 (IL-6), stimulates the metastasis of several neoplasms. An association of its serum level and the single nucleotide polymorphism (SNP) rs1800795 with neuroblastoma (NB) has been reported in American and Italian cohorts. This study was to clarify whether the same association exists in Chinese children.</p><p><b>Methods</b>A total of 130 NB patients, with 77 boys (59%), 53 girls (41%), mean age 41 ± 5 months, were assigned to two groups: high risk (HR) versus intermediate-low risk (non-HR), and 50 healthy children were randomly selected as the age- and gender-matched controls. Peripheral blood samples were analyzed to determine serum IL-6 level using enzyme linked immunosorbent assay and rs1800795 SNPs phenotype using polymerase chain reaction and gene sequencing.</p><p><b>Results</b>There were 87 NB patients in the HR group and 43 NB patients in the non-HR group. A comparison of allele and genotype frequencies of the rs1800795 polymorphism between patients and controls found no association with NB risk (P > 0.05). The frequency of GG+GC genotype was higher in HR-NB patients than in non-HR-NB patients (64.4% vs. 48.8%, P = 0.02), and serum IL-6 level was much higher in HR-NB patients with GG+GC genotype than in HR-NB patients with CC genotype (4.36 ± 1.1 pg/ml vs. 1.83 ± 0.5 pg/ml; P = 0.02), but not in Non-HR-NB patients.</p><p><b>Conclusions</b>The polymorphism rs1800795 is associated with serum IL-6 level and level of NB risk. GG genotype might indicate that the tumor is highly malignant (prone to metastasis) and associated with poor prognosis.</p>

Transcription factors analysis of subchondral bone in early experimental osteoarthritis based on gene expression profiles / 中国骨伤

<p><b>OBJECTIVE</b>To identify the master transcription factors (TF) that might be responsible for the gene expression alteration of OA.</p><p><b>METHODS</b>Raw expression data for rat OA model(GSE30322) was downloaded from NCBI GEO database. Microarray data analysis for rat and human was carried out separately using functions from limma packagein R, gene expression was considered as significantly changed between conditions if adjusted-value<0.05 and the absolute value of fold change>=2. iRegulon was applied to differentially up-regulated and down-regulated genes in OA separately.</p><p><b>RESULTS</b>(1)15 TFs, including FOXN4, NANOS1, E2F6, RAD21, MECOM, ETS1, MEF2A, POU2F3, BRCA1, GATA3, ZNF706, ZBTB33, SUZ12, DBP and SETDB1, were identified as the potential master TFs of up-regulated DEGs with statistical significance. (2)12 TFs, including ARID3A, YY1, RDBP, ATF1, CRX, TAF1, XBP1, SOX3, E2F4, PGR, TIMM8A and HOXA2, were identified as the potential master TFs of down-regulated DEGs with statistical significance.</p><p><b>CONCLUSIONS</b>The newly identified TFs maybe play important roles in pathogenesis of early experimental osteoarthritis, and our study provides new diagnostic markers or therapeutic targets for OA.</p>

Cloning and expression analysis of a zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein encoding gene in Dendrobium officinale / 中国中药杂志

The zinc-regulated transporters (ZRT), iron-regulated transporter (IRT)-like protein (ZIP) plays an important role in the growth and development of plant. In this study, a full length cDNA of ZIP encoding gene, designed as DoZIP1 (GenBank accession KJ946203), was identified from Dendrobium officinale using RT-PCR and RACE. Bioinformatics analysis showed that DoZIP1 consisted of a 1,056 bp open reading frame (ORF) encoded a 351-aa protein with a molecular weight of 37.57 kDa and an isoelectric point (pI) of 6.09. The deduced DoZIP1 protein contained the conserved ZIP domain, and its secondary structure was composed of 50.71% alpha helix, 11.11% extended strand, 36.18% random coil, and beta turn 1.99%. DoZIP1 protein exhibited a signal peptide and eight transmembrane domains, presumably locating in cell membrane. The amino acid sequence had high homology with ZIP proteins from Arabidopsis, alfalfa and rice. A phylogenetic tree analysis demonstrated that DoZIP1 was closely related to AtZIP10 and OsZIP3, and they were clustered into one clade. Real time quantitative PCR analysis demonstrated that the transcription level of DoZIP1 in D. officinale roots was the highest (4.19 fold higher than that of stems), followed by that of leaves (1.12 fold). Molecular characters of DoZIP1 will be useful for further functional determination of the gene involving in the growth and development of D. officinale.

Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinale / 药学学报

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.

Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein / 药学学报

HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.

Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale / 药学学报

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

Observation and prevention of toxicity of super high dose of cyclophosphamide chemotherapy scheme for treating children with high risk neuroblastoma / 中华实用儿科临床杂志

Objective To observe a variety of the toxicity,and the incidence of super-high-dose cyclophosphamide as a main chemotherapy scheme for treating children with high risk neuroblastoma,and to summarize the prevention and cure of the disease.Methods The high risk neuroblastoma patients who were diagnosed in Hematologic Tumor of Beijing Children's Hospital Affiliated to Capital Medical University were selected.And they were received super high dose of cyclophosphamide mainly chemotherapy scheme-CAV (cyclophosphamide,adriamycin and vincristine) more than 2 courses according to BCH-NB-HR(Beijing Children's Hospital-Neuroblastoma-High Risk) scheme.Their clinical symptoms,signs,blood routine and blood biochemistry in 1-2-course of treatment were retrospectively analyzed and summarized.According to WHO anti-cancer drugs in acute and subacute toxicity reaction grading standards,the data were analyzed.Results There were 33 children consistent with the criterion of research,who received 66 times of super-high-dose cyclophosphamide as a main chemotherapy scheme.All of the patients had bone marrow suppression,granulocyte generated Ⅳ grade toxicity 56 (84.8％),white blood cell (＜ 1.0 × 109/L) Ⅳ grade toxicity was 56 (84.8％),and blood platelet(＜25 x 109/L) Ⅳ grade toxicity was 19(28.8％).Hemoglobin(＜65 g/L) Ⅳ grade toxicity was 4 (6.06％).The digestive system toxicity including nausea and vomiting toxicity (100.0％).Urogenital system toxicity:Hemorrhagic cystitis only was 1 (1.52％) ;Cardiac Ⅰ-Ⅲ grade toxicity was 3(4.55％).But skin and nervous system toxicity was not observed.No patients died from chemotherapy.Conclusions There is a high incidence of toxicity with super-high-dose of cyclophosphamide as a main chemotherapy scheme,and the highest incidence ineludes bone marrow suppression,digestive tract response and secondary infection.But side effects like hemorrhagic cys-titis,liver,kidney and cardiac toxicity are not significant.Super-high-dose cyclophosphamide chemotherapy scheme is clinically safe under the perfect supportive treatment and rigorous monitoring.

Rapid determination of four components in Guizhi Fuling capsule with online two-dimensional liquid chromatography / 中国中药杂志

To establish the online two-dimensional liquid chromatography by using double gradient liquid chromatography system and UV detector, in order to simultaneously determine the content of paeoniflorin, paenol, amygdaloside and cinnamic acid. A pump of the two-dimensional liquid chromatography was adopted as the one-dimensional separation pump. C18 (3.0 mm x 150 mm, 3 microm) was used as the analytical column, with acetonitrile as the organic phase and 0.08% phosphoric acid + 0.08% triethylamine as the aqueous phase for gradient elution at the flow rate of 0.5 mL x min(-1). Another pump of the two-dimensional liquid chromatography was adopted as the two-dimensional separation pump. PAII C18 was used as the analytical column, with acetonitrile as the organic phase and 20 mmol, pH 3.0 monopotassium phosphate as the aqueous phase for gradient elution at the flow rate of 0.8 mL x min(-1). The detection wavelengths were set at 218, 230, 275 nm by using wavelength time-switching program. The linearity range of paeoniflorin, amygdaloside, paeonol and cinnamic acid were 5.55-222 (r = 0.999 7), 6.6-264 (r = 0.999 8), 3.3-132 (r = 0.999 5) and 0.315-12.6 mg x L(-1) (r = 0.999 7), respectively. The average recoveries of the four components were between 96.12% and 103.9%. The experiment proved that this method was so rapid and accurate in determination results that it could be used for evaluating drug quality.

Safety and efficacy of percutaneous transcatheter closure of atrial septal defect under transesophageal echocardiography guidance in children / 中华心血管病杂志

<p><b>OBJECTIVE</b>To assess the safety and effectiveness of percutaneous transcatheter closure of atrial septal defect (ASD) under transesophageal echocardiography (TEE) guidance in children.</p><p><b>METHODS</b>The study included 20 cases of patients with ASD. The patients were (4.2 ± 1.2) years old and the mean body weights were (18.2 ± 4.2) kg. The diameter of ASD before closure was (13.4 ± 3.3) mm . All procedures were guided under TEE. Procedure success was evaluated by TEE immediately after procedure.</p><p><b>RESULTS</b>Closure devices were successfully implanted in all 20 patients under TEE guidance. The diameter of closure devices was 14-26 mm. There were no procedure related complications. The ventilation time was (2.9 ± 0.8)h and the hospitalization time was (3.2 ± 0.7) days.</p><p><b>CONCLUSION</b>TEE guided percutaneous transcatheter closure is safe and effective for patients with ASD and avoids the radiation damages.</p>

Isolation, idetification and anti-HIV-1 integrase activity of culturable endophytic fungi from Tibetan medicinal plant Phlomis younghusbandii Mukerjee / 药学学报

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale / 药学学报

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

Cloning and expression analysis of a calcium-dependent protein kinase gene in Dendrobium officinale in response to mycorrhizal fungal infection / 药学学报

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.

Molecular characterization of a mitogen-activated protein kinase gene DoMPK1 in Dendrobium officinale / 药学学报

The mitogen-activated protein kinase (MAPK) cascade, composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza (OM) symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 (GenBank accession No. JX297594), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point (pI) of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain (39-325) and MAP kinase signature (77-177). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous (71%-85%) to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.

N-myc gene copies in children with neuroblastoma and its clinical significance / 中国实验血液学杂志

This study was purposed to analyze the relation of N-myc gene copy number with clinical staging, pathological types and tumor biological factors in children with neuroblastoma (NB), and to investigate the influence of chemotherapy on N-myc gene expression and explore the relationship of N-myc gene copies with prognosis of NB children. The newly diagnosed children with NB from 1 March 2007 to 31 January 2011 were enrolled in this study. The treatment was carried out by BCH-NB-2007 based on Hongkong NB-07 protocol, and the patients were follow up to 31 January 2012. The N-myc gene in NB children was detected by FISH. According to number of N-myc gene copies, the NB children were divided into 3 groups. A group (N-myc gene negative) had less than 2 copies, B group (N-myc gene gains) had 3 to 9 copies, and C group (N-myc amplification) had more than 10 copies. The results showed that the N-myc gene expression in 58 cases of NB was observed. There were 36 males and 22 females. NB children aged from 6.5 to 138 months (median age 47.5 months), all patients were followed up for 11 - 57 months with an average of 31.5 months. INSS stages I-IV were 1, 5, 8 and 44 cases, respectively. Twenty-five cases had primary post mediastinal tumor, thirty-three cases had retroperitoneal and pelvic tumor, three of which also companied with post mediastinal tumor. Thirty-five cases had bone metastasis (60.3%), thirty-two cases had bone marrow metastasis (55%). Of the 54 patients with fully known biologic features, seventeen cases had ganglioneuroblastoma, thirty-seven cases had neuroblastoma (15 displayed differentiated, 7 poorly differentiated or undifferentiated, 15 with pathological changes after chemotherapy), four cases had bone marrow metastasis only detected by bone marrow biopsy. Eleven cases had N-myc gene negative, forty-three had N-myc gains, four had N-myc amplification. The average copy number of N-myc gene copies in 58 cases was 5.96 ± 7.81 in which 28 children were non chemotherapy cases, their average copy number was 4.00 ± 1.88, thirty cases out of 58 cases received preoperation chemotherapy (chemotherapy group), and their average copy number was 7.80 ± 10.46, the difference is significant (P = 0.064). The clinic stage, the location of primary tumor, pathological classification, urine VMA and serum neurogenic specific enolase had no effects on the N-myc gene expression, but the serum LDH level had influence (P < 0.01). Single factor Kaplan-Meier analysis showed that the number of N-myc gene copies in NB patients were closely related with the poor prognosis. The more copies of N-myc gene, the more poor prognosis, the difference is statistically significant (P < 0.05). It is concluded that the number of N-myc gene copies correlates with the rapid growth of NB and its poor prognosis, detecting the N-myc amplification can help to estimate the prognosis and decide the program of treatment. Serum LDH, which correlated with the rapid growth of NB, had effect on the N-myc gene expression and is closely related with the poor prognosis of NB.

Thiopurine S-methyltransferase gene sequence analysis of ALL children severely intolerant to 6-mercaptopurine / 中国实验血液学杂志

This study was aimed to analyze the thiopurine S-methyltransferase (TPMT) gene sequence in acute lymphoblastic leukemia (ALL) children severely intolerant to 6-mercaptopurine (6-MP) and to investigate the causes resulting in tolerance difference to 6-MP in ALL children so as to provide evidence for safe and rational use of 6-MP. The adverse reactions of drug was evaluated in ALL children treated with BCH-2003-ALL chemotherapeutic protocol during 2004-10-1 to 2007-9-30 according to NCI-CTC V2.0. The TPMT gene sequences of ALL children with 3-4 grade of severe toxicity during the maintenance therapy were analyzed by PCR and direct DNA sequencing. To assure the accuracy of sequencing, the 738 bp fragment of coding region in TPMT gene (NM_000367) was divided into 3 subfragments and bidirectionally sequenced. The results indicated that among 133 ALL children, 61 were severely intolerant to 6-MP. The direct DNA sequencing showed that among 59 patients (excluding 2 cases without RNA samples), the simple myelotoxicity was found in 37 cases, hepato-myelotoxicity was observed in 9 cases, hepatotoxicity along appeared in 12 cases, 1 case showed skin rash. Out of 59 ALL children, the C474T mutation was found in 57 cases, with mutation rate 96.6%, including 21 cases with heterozygous mutation and 36 cases with homozygosis mutation. The TPMT gene sequencing of 10 cases tolerant to 6-MP indicated that C474T mutation was detected in 8 cases which was homozygous mutation. It is concluded that the C474T mutation in 738 bp fragment of coding region in TPMT gene is very frequent, but it is not related with tolerance to 6-MP, suggesting that severe intolerance to 6-MP in ALL children may be not related with the mutation of coding region in TPMT gene.