The Effect of Novel Phosphodiesterase 4 Inhibitor ZL-n-91 to the Proliferation of Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the inhibitory effects of novel phosphodiesterase 4 inhibitor ZL-n-91 to the proliferation of leukemia cells L1210 and K562.@*METHODS@#CCK-8 method was used to detect the effect of ZL-n-91 to the proliferation of L1210 and K562 cells, and the proliferation rate, IC@*RESULTS@#ZL-n-91 showed a significant inhibitory effect to the proliferation of leukemia cells L1210 and K562 in a dose-dependent manner (P<0.001). After treated by ZL-n-91, the leukemia cells L1210 and K562 in the S-phase in cell cycle decreased significantly compared with those in control group (P<0.01). The apoptosis of leukemia cells L1210 and K562 could be induced by ZL-n-91 (P<0.001), and the expression level of apoptosis related protein BAX significantly increased. In the animal experiment, the result showed that ZL-n-91 could significantly inhibit the growth of subcutaneously transplantation tumor (P<0.05).@*CONCLUSION@#The novel phosphodiesterase 4 inhibitor ZL-n-91 can effectively inhibit the proliferation of leukemia cells L1210 and K562, which has the potential of anti-leukemia drug development.

Effect of Culture Supernatant Derived from Trichophyton Rubrum Grown in the Nail Medium on the Innate Immunity-related Molecules of HaCaT / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively.</p><p><b>METHODS</b>The culture supernatants of two strains (T1a, T XHB ) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test.</p><p><b>RESULTS</b>The T. rubrum strains (T1a and T XHB ) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h.</p><p><b>CONCLUSION</b>The culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.</p>

Epidemiological analysis of childhood asthma in Yichang City, China / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the prevalence, epidemiological characteristics, and risk factors for childhood asthma in Yichang City, China and to collect evidence for the early diagnosis and preventive treatment of asthma.</p><p><b>METHODS</b>Preliminary screening questionnaires were distributed to more than 90% of children in 5 kindergartens, 10 primary and secondary schools, and 5 communities in Yichang City to detect children with suspected asthma. These surveyed children were selected by cluster random sampling. A further questionnaire survey was conducted for suspected cases. Meanwhile, a similar number of sex- and age-matched non-asthmatic children were selected for the case-control study. Information from returned questionnaires was entered into a database for statistical analysis.</p><p><b>RESULTS</b>A total of 11 000 questionnaires were distributed, and 10 456 (95.1%) questionnaires were returned. The prevalence rate of asthma among children in Yichang was 3.47%, significantly higher in boys than in girls (P<0.05). A total of 107 out of 363 children with asthma had a history of drug allergy, and 152 cases had a family history of allergy. The majority of asthmatic children had irregular onset-prone seasons and hours. Respiratory tract infections were the most common trigger of asthma attacks, accounting for 93.1% of all onsets; family history of allergy, history of early use of antibiotics, history of housing renovation, and history of passive smoking were the major risk factors for asthma.</p><p><b>CONCLUSIONS</b>Prevention of respiratory tract infections may reduce the frequency of asthma attacks; reducing the use of antibiotics during early childhood, decreasing the frequency of housing renovation, and advocating for smoking cessation among parents have preventive effects on asthma.</p>

Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.</p><p><b>METHODS</b>HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.</p><p><b>RESULTS</b>HaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.</p><p><b>CONCLUSION</b>The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.</p>

Effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes cultured with high glucose / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Diabetic cardiomyopathy is the major cause of morbidity and mortality in diabetic patients. Oxidative stress plays an important role in diabetic cardiomyopathy. This study aimed to investigate the effects of adiponectin on oxidative stress and apoptosis in human cardiac myocytes (HCM) cultured with high glucose.</p><p><b>METHODS</b>The cells were assigned to three group: control group, high glucose group and high glucose plus adiponectin group. After culture for 24, 48, 72 hours, oxidative stress was evaluated by detecting levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the supernatant of culture media. The expression of p66Shc and Heme oxygenase-1 (HO-1) was detected by real-time polymerase chain reaction (PCR). Flow cytometry was designed to observe and detect cellular apoptosis.</p><p><b>RESULTS</b>Our findings showed significant increase in MDA levels and decrease in SOD activity in the high glucose group compared with the control group (P < 0.05). However, MDA levels were significantly decreased and SOD activity was significantly increased in the adiponectin group compared with those in the high-glucose group (P < 0.05). The mRNA expression of HO-1 in the high glucose group was significantly increased in a time-dependent manner compared with that in the control group (P < 0.05). Adiponectin further increased the mRNA expression of HO-1 induced by high glucose in a time-dependent manner (P < 0.05).The expression of p66Shc was significantly increased in high glucose group compared with that in the control group (P < 0.05). Adiponectin significantly suppressed the upregulation of p66Shc induced by high glucose (P < 0.05). The apoptotic rate of cardiomyocytes was significantly increased in the high glucose group compared with that in the control group while the apoptotic rate in the adiponectin group was remarkably declined in comparison with that in the high glucose group.</p><p><b>CONCLUSION</b>Adiponectin reduces high glucose-induced oxidative stress and apoptosis and plays a protective role in myocardial cells by upregulating the HO-1 expression and downregulating p66Shc expression.</p>

The immune response of human keratinocytes to Trichophyton rubrum conidia is partially mediated by toll-like receptor-2, 4, dectin-1 and cytokines / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of Trichophyton rubrum exposure on the expressions of toll-like receptor-2 (TLR-2), TLR-4 and dendritic cell associated C-type lectin-1 (Dectin-1) and cytokine secretions in human keratinocytes cell line HaCaT.</p><p><b>METHODS</b>The mRNA of TLR-2,4, and dectin-1 in the HaCaT co-cultured with the conidia of Trichophyton rubrum conidia for 24 h was measured with real-time PCR. The mean fluorescence intensity and the percentage of cells positive for TLR-2, 4, and dectin-1 was detected during the co-culture using flow cytometry. The cytokine secretion profiles in the cell culture supernatant was analyzed using a cytokine antibody array.</p><p><b>RESULTS</b>The TLR-2,4, and dectin-1 mRNA expressions, mean fluorescence intensity and percentage of positive cells for TLR-2,4, and dectin-1 all increased in HaCaT cells in response to Trichophyton rubrum conidia exposure. The results of cytokine antibody array demonstrated obviously increased secretions of IL-8, I-309, IFN-γ, IL-6, and IL-13 in the culture supernatant of HaCaT cells in response to Trichophyton rubrum exposure.</p><p><b>CONCLUSION</b>The immune responses and immunological recognition of human keratinocytes to Trichophyton rubrum conidia are partially mediated by up-regulating the expressions of TLR-2, TLR-4 and dectin-1 and secretions of multiple cytokines.</p>

Correlation between MECP2 genotype and phenotype in Chinese patients with Rett syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Rett syndrome (RTT) is a neurodevelopmental disorder that represents one of the most common genetic causes of mental retardation in girls. The aim of this study was to investigate the correlation between MECP2 genotype and phenotype and thereby not only to provide assistance for clinical care, but also facilitate clinical genetic counseling.</p><p><b>METHOD</b>Individual phenotype characteristic and clinical severity of 126 children with RTT diagnosed by molecular genetic methods were evaluated by using scales of Kerr et al and Scala et al. Statistical package SPSS 12.0 was used for analyses of data. Since the majority of the data were not normally distributed, non-parametric tests were used. The Kruskal-Wallis test/Wilcoxon Mann-Whitney test was employed to compare total severity phenotype scores. The Fisher exact test was used for comparing rates. Statistical significance was set at P < 0.05.</p><p><b>RESULT</b>There were no significant differences in the average overall scores for RTT patients with mutations in the region of methyl-CpG-binding domain (MBD) compared with those mutations in the transcription repression domain (TRD) and C terminal segment (CTS), also patients with nonsense mutations compared with missense mutations, frameshift mutations and large deletions (P > 0.05). The RTT patients with nonsense mutations located in the region of MBD have more severe phenotype than those with missense mutations in the same region (P = 0.016). Among p.T158M, p.R168X, c.806delG and p.R255X, there were no significant differences in the average overall scores (P > 0.05), but there were significant differences in language skill (P = 0.028) and in language impairment rate at different level (P = 0.019).</p><p><b>CONCLUSION</b>There are relationships between MECP2 genotype and phenotype:the RTT patients with nonsense mutations located in MBD tend to develop more severe phenotype;there are significant differences in language skill and language impairment rate in the groups with p.T158M, p.R168X, c.806del and p.R255X, which had higher frequency in children below five-years of age and the p.R168X present with most severe impairment.</p>

Analysis of the parental origin of de novo MECP2 mutations and X chromosome inactivation in fifteen sporadic cases with Rett syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Rett syndrome (RTT) is a neurodevelopmental disorder occurring almost exclusively in females as sporadic cases due to de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Familial cases of RTT are rare and are due to X-chromosomal inheritance from a carrier mother. Recently, DNA mutations in the MECP2 have been detected in approximately 84.7% of patients with RTT in China. To explain the sex-limited expression of RTT, it has been suggested that de novo X-linked mutations occur exclusively in male germ cells resulting therefore only in affected daughters. To test this hypothesis, we have analyzed the parental origin of mutations and the XCI status in 15 sporadic cases with RTT due to MECP2 molecular defects.</p><p><b>METHODS</b>Allele-specific PCR was performed to amplify a fragment including the position of the mutation. The allele-specific PCR products were sequenced to determine which haplotype contained the mutation. It was then possible to determine the parent of origin by genotyping the single nucleotide polymorphism (SNP) in the parents. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes by analyzing CAG repeat polymorphism in the androgen receptor gene (AR).</p><p><b>RESULTS</b>Except for 2 cases who had a frameshift mutation; all the remaining 13 cases had a C-->T transition mutation. Paternal origin has been determined in all cases with the C-->T transition mutation. For the two frameshift mutations, paternal origin has been determined in one case and maternal origin in the other. The frequency of male germ-line transmission in mutations is 93.3%. Except for 2 cases who were homozygotic at the AR locus, of the remaining 13 cases, 8 cases had a random XCI pattern; the other five cases had a skewed XCI pattern and they favor expression of the maternal origin allele.</p><p><b>CONCLUSION</b>De novo mutations in sporadic RTT occur almost exclusively on the paternally derived X chromosome and that this is most probably the cause for the high female: male ratio observed in sporadic cases with RTT. Random XCI was the main XCI pattern in sporadic RTT patients. The priority inactive X chromosome was mainly of paternal origin.</p>

Application of long range polymerase chain reaction and DNA direct sequencing in diagnosis of Rett syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Rett syndrome (RTT, MIM 312750) is a progressive neurodevelopmental disorder that affects females almost exclusively, caused by mutations in MECP2 gene on chromosome Xq28, with symptoms such as autism, severe mental deficiency, deceleration of head growth, ataxia, loss of purposeful hand function and characteristic stereotypic hand movements. Over 80% MECP2 mutations located in the exon 3 and exon 4 were confirmed by our work and large-scale studies. RTT is defined based on clinical presentation. It is difficult to diagnose in the early life without definite biochemical abnormality, but genetic test is helpful for this. The aim of this study was to investigate the feasibility and clinical significance of applying long range polymerase chain reaction (PCR) to RTT diagnosis and establish a simple, economic, efficient method of genetic diagnosis.</p><p><b>METHOD</b>Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of each patient. Long range polymerase chain reaction(PCR)and DNA direct sequencing were employed to analyze the exon 3 and 4 of MECP2 gene simultaneity in 40 patients with RTT. The PCR products were checked by using 1.5% agarose gel.</p><p><b>RESULT</b>In total, 18 different MECP2 mutations were identified in 33 of the 40 diagnosed sporadic female patients with RTT. Missense mutations were 16, followed by 14 nonsense mutations and 3 deletions. The 314 base pairs large deletion was identified. The p. T158M mutation (21%, 7/33) was the most common, followed in order of frequency by p. R255X (12%, 4/33), p. R168X and p. R106W (9%, 3/33) respectively, p. R270X and p. Y141X (6%, 2/33) respectively, p. R133C, p. D156H, p. P157L, p. P225R, p. Q244X, p. Q262X, p. R294X, p. R306C, P322L, c. 1005del G, c.1005-1318del 314 bp and c.1127-1179del 53 bp (3%, 1/33), respectively.</p><p><b>CONCLUSION</b>Long range PCR is a simple, economic, quick, precise method of genetic diagnosis and was able to find 83% MECP2 gene mutations in RTT patients in this study. It is helpful for RTT clinical diagnosis in early stage. On the other hand, it may detect recurrent mutations and large deletions at the same time.</p>

Content Determination of Astragaloside Ⅳ in Qingshen Jianfei Tablets by HPLC-ELSD / 中国中医药信息杂志

Objective To establish a HPLC-ELSD method for determining the content of astragaloside Ⅳ in Qingshen Jianfei Tablets. Methods Stationary phase was C_(18) column (4.6 mm?250 mm, 5 ?m), mobile phase was acetonitrice-water (35 : 65). The evaporation temperature was 120 ℃, the pulverization temperatire was 80 ℃, the flow rate was 1.6 mL/min and column temperature was 35 ℃. Results The standard curve was linear within the range of 1.03～8.24 ?g, r=0.9999. The average recovery was 99.35% with RSD = 1.36% (n = 6). Conclusion The established method is simple and accurate, with good reproducibility and high precision, and suitable for the determination of astragaloside Ⅳ in Qingshen Jianfei Tablets.

X chromosome inactivation patterns in patients with Rett syndrome and their mothers and the parental origin of the priority inactive X chromosome / 中华儿科杂志

<p><b>OBJECTIVE</b>Rett syndrome (RTT) is a severe childhood neurodevelopmental disorder mainly affecting females. The pathogenic gene is located at Xq28, which codes for the methyl-CpG-binding protein 2. MECP2 gene is affected by X chromosome inactivation (XCI). The different XCI patterns of females could affect the expression ratios of pathogenic gene, causing changes in clinical symptoms. In order to understand the XCI patterns in RTT patients and the relationship between XCI pattern, genotype and phenotype, the XCI patterns in patients with RTT and their mothers, the parental origin of the priority inactive X chromosome in RTT, and the relations of XCI patterns with genotype and phenotype in RTT cases were analyzed.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood of 55 cases with RTT (52 with MECP2 mutations, 3 without mutations), 53 mothers of RTT cases and 48 normal female controls. DNA was digested with methylation sensitive restriction endonuclease Hpa II. Then the undigested and digested DNAs were amplified via PCR for the first exon of human androgen receptor (AR) gene. PCR products were analyzed by Genescan.</p><p><b>RESULTS</b>The heterozygotic rates of AR gene were 82%, 77% and 83% in RTT patients, mothers and controls, respectively. XCI distribution pattern of RTT was different from that of the mothers and control, P < 0.05. More mothers and controls than RTT patients were in the area of XCI 50:50 - 59:41. The differences between them were statistically significant (P < 0.05). No significant difference in XCI distribution patterns between mothers and the control groups was found (P > 0.05). Non-random XCI rates in the areas of XCI >or= 65:35 and >or= 80:20 were 53.35% and 17.8%, respectively, in RTT patients, compared with the mothers group (36.6%, 7.3%) and control group (35%, 10%), it was higher in RTT patients, but the difference was not statistically significant (P > 0.05). In 18 of 21 cases with XCI >or= 65:35, the priority inactive X chromosome was of paternal origin (85.7%). Variable XCI patterns were observed in the same gene mutation patients. The highly skewed XCI as well as the random XCI were found in patients with mild, severe and typical phenotype. The rate of highly skewed XCI in atypical patients was higher than that in typical RTT patients. The rate of highly skewed XCI in T158M was higher than the other type mutations. No highly skewed XCI was observed in cases with R133C mutation.</p><p><b>CONCLUSION</b>The XCI distribution pattern of RTT patients was different from that of RTT mother and control groups. There was no significant difference in XCI distribution patterns between mothers and the control groups. It was not a main genetic pattern in RTT that mothers as the carriers to transmit the pathogenic gene to the patients. Non-random XCI was not the main XCI pattern in RTT patients. The priority inactive X chromosome was mainly of paternal origin. XCI could modify the clinical phenotype of RTT, but had limitations in explaining all the phenotypes manifested in RTT cases.</p>

MECP2 gene mutations in twenty-six cases with atypical Rett syndrome / 中华儿科杂志

<p><b>OBJECTIVE</b>Rett syndrome (RTT) is an X-linked progressive neurodeveopmental disorder that almost exclusively affects girls, and is one of the most common causes of mental retardation in females, with an estimated prevalence of approximately 1 in 10,000 - 15,000 female individuals. Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) gene, located on chromosome Xq28, have been found to be a cause of RS. A lot of mutations have been reported to be related to RS recently. Mutations are found in 70% - 85% of patients with classical RTT and in less than 50% of patients with atypical RS. Up to now, RTT is diagnosed based on a consistent counseling for clinical features and the established diagnostic criteria. The present study aimed to investigate frequency and type of mutation of MECP2 gene and if hot spot of mutation exits in patients with atypical RTT and find out the relationship between genotype and phenotype.</p><p><b>METHODS</b>A systematic analysis of the entire coding region of MECP2 in 26 unrelated patients with atypical RTT was performed by polymerase chain reaction (PCR) and direct sequencing. Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of each patient. PCR amplification products were checked by 2% agarose gel electrophoresis and were subsequently sequenced with ABI 3730 Automated DNA Sequencer with both the forward and reverse primers. Mutational analyses were performed using normal human genomic MECP2 sequence as a reference (GenBank accession NO.AF030876).</p><p><b>RESULTS</b>Seven mutations were identified in 12 of 26 patients. Most of the mutations were missense mutation; c.397C > T (R133C) was found in 3 of 26 patients; c.473C > T (T158M) and c.916C > T (R306C) were found in 2 of 26 patients, respectively; c.397A > G (R133H) and c.1005G > A (R335C) were found in 1 of 26 patients, respectively. One base pair deletion mutation (806delG) resulting in frameshift was found in 2 of 26 patients, and 1 base pair transversion at splice accept-site (IVS3-2A > T).</p><p><b>CONCLUSION</b>The results of this study indicated that c.397C > T (R133C), c.473C > T (T158M) and c.916C > T (R306C) were hot spot mutations in MECP2 gene of patients with atypical RTT. There was some relationship between genotype and phenotype.</p>