ABSTRACT
Objective To obtain a full-length cDNA of squalene synthase gene from Hedera helix (HhSS) by cloning technique, and to carry out bioinformatics analysis and expression analysis. Methods Primers were designed based on H. helix transcriptome data, and HhSS was cloned by using RACE technologies. DNAMAN, PROTPARAM, TMHMM, PSORT, ScanProsite, SOPMA, and SWISS-MODEL were used for analysis of sequence and physical and chemical properties and domain of encoded protein. The relative expression of HhSS was detected by qRT-PCR. Results The cDNA sequence of HhSS (GenBank accession number: KX056078) was 1 889 bp which obtained by RACE cloning. It contained an ORF from 248 bp to 1 477 bp and a 5'UTR with 247 bp and a 3'UTR with 412 bp. It encoded a 409-amino-acid protein with a molecular weight of 46 800and an isoelectric point (pI) of 5.68. The HhSS protein had the characteristic domain and transmembrane region of plant SS protein and closer relationship with Eleutherococcus senticosus, Panax ginseng et al. The qRT-PCR results indicated that it had positive correlation between relative expressing level of HhSS gene and contents of saponins in H. helix leaves. Conclusion The cloning and expression analysis results of HhSS provide a theoretical and technical basis for elucidating the role of HhSS in saponins biosynthetic pathway and metabolic regulation.
ABSTRACT
Objective: This study detected the contents of four saponins (hederacoside C, hederacoside D, hederacoside D, α-hederin) from different parts of annual cutting Hedera helix in one year growth cycle and analyzed the dynamic accumulation, in order to provide theoretical basis to harvest Hedera helix L. as medicinal raw materials reasonably. Methods: This study designed a reasonable method of collecting materials and detected the contents of saponins by HPLC. Results: The contents of saponins in different parts were as following order: mature leaf > new leaf > stem > root, and four saponins in different parts had different dynamic accumulations in one year growth cycle. Conclusion: The leaf and stem are the reasonable collecting parts of raw materials which have higher medicinal quality. The reasonable collecting time of leaf is the growth stage in March and stem is from March to November before the beginning of dormancy.
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Objective: Through establishing the HPLC-UV fingerprint of saponins from the plants of varieties of Hedera helix L., using cluster and principal component analysis methods to do statistical analysis and combining with the determination of the calibration compound to provide a reference for the medicinal quality evaluation on the plants of varieties of Hedera helix L. Methods: The chromatographic separation was performed on Unitary C18 column (250 mm × 4.6 mm, 3 μm); The mobile phase was a mixture of acetonitrile-0.2% phosphoric acid solution; The elution mode was binary high pressure gradient system; The detection wavelength was 205 nm; The flow rate was 1 mL/min; The injection volume was 20 μL and the column temperature was 25 ℃. Results: Using the similarity evaluation system for chromatographic fingerprint of Chinese materia medica to calibrate 12 common peaks of 17 samples of varieties of Hedera helix L. and calculate their similarity between 0.715-0.992; The preliminary results is that the medicinal quality of ivy is better than the other varieties of varieties of Hedera helix L. by analysis of cluster and principal component. Conclusion: The HPLC-UV fingerprint can be used to screen the high quality raw materials of varieties of Hedera helix L. and do the quality evaluation and control, in the meantime, it can be used to provide a new reference of studying on the medicinal quality evaluation for the plants of varieties of Hedera helix L.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the relevance between the promoter methylation status of Notch1 gene and the invasive ductal carcinoma and ductal hyperplastic lesions of the breast.</p><p><b>METHODS</b>Methylation status of Notch1 gene in human breast invasive ductal carcinoma (IDC, n = 89), ductal carcinoma in situ (DCIS, n = 20), atypical ductal hyperplasia (ADH, n = 11) and usual ductal hyperplasia (UDH, n = 20) were quantitatively evaluated by MALDI-TOF MS. The expression of Notch1 protein was detected by immunohistochemical stain (SP method).</p><p><b>RESULTS</b>Positive expression rates of Notch1 protein in IDC and DCIS were 91.0% (81/89) and 75.0% (15/20), respectively, which were significantly higher than those of ADH (4/11) and UDH (30.0%, 6/20;P < 0.05). Notch1 protein expression was correlated significantly with lymph node metastasis, pathological grades and TNM stages of IDC. The mean methylation levels of Notch1 gene at CpG_3, CpG_4.5 and CpG_8 significantly decreased in IDC group compared with those of DCIS, ADH and UDH groups (P < 0.0083). In breast carcinomas, the mean methylation rates of Notch1 gene at CpG_4.5, CpG_10.11, and CpG_14.15.16 loci in cases with axillary node metastasis were significantly lower than those without axillary node metastasis (P < 0.05); and the methylation rates at CpG_14.15.16 and CpG_18 loci in stage Iwere lower than that in stage II, further lower than that in stage III (P < 0.05); and that in CpG_1.2, CpG_12.13 loci in grade I (highly-differentiated group) were higher than that in grade II (moderate-differentiated group) and grade III (poorly-differentiated group) (P < 0.05); and the methylation rates at CpG_3, CpG_8 and CpG_14.15.16 loci in ER(+) PR(+) HER2(-) group were lower than that in ER(-) PR(-) HER2(+) group (P < 0.05).</p><p><b>CONCLUSIONS</b>There is an overall hypomethylation of Notch1 gene in breast invasive ductal carcinomas with corresponding over-expression of Notch1 protein. This inverse correlation show that the alteration of protein expression result from hypomethylation oncogene Notch1, and this change may have important significance in breast tumorigenesis and the development. Specific hypomethylation at CpG_3, CpG_ 4.5 and CpG_8 loci of Notch1 gene may play a role in the pathogenesis of breast carcinoma, suggesting the progression and/or malignant transformation from benign glandular lesions of the breast.</p>