Discovery of tetrandrine derivatives as tumor migration, invasion and angiogenesis inhibitors.

Metastatic progression of cancer is a complex and clinically daunting process, with migration, invasion and angiogenesis being the key features. Tetrandrine (TET) is a typical dibenzylisoquinoline alkaloid with promising anti-tumor activity. In our previous work, a number of TET derivatives were designed and synthesized with obvious anti-proliferation activities against cancer cells, however, the anti-metastatic effects of these compounds were not evaluated. In the current investigation, five TET derivatives (8, 18, 32, 71, and 72) with pronounced anti-proliferative activities (IC50 values of 1.00, 1.91, 3.43, 3.78, and 1.93 µM, respectively) against human umbilical vein endothelial cells (HUVECs) were screened out. Scratch assays showed that these compounds significantly suppressed the migration of HUVECs and induced their apoptosis. Among them, derivatives 8 and 72 obviously inhibited the proliferation, colony formation and invasion of HCT-15 cells. Tube formation assays revealed that 4 µM of 8 or 72 remarkably inhibited the tube forming capacity of HUVECs. Moreover, 8 and 72 surpressed the formation of filopodia in HUVECs and severely impaired their motility. Both compounds effectively inhibited the angiogenesis in the zebrafish model with low toxicities in vivo. These results indicated that TET derivatives 8 and 72 are promising anti-metastatic inhibitors.

G-protein beta 3 subunit polymorphisms and essential hypertension: a case-control association study in northern Han Chinese.

OBJECTIVE: To explore the association between the three polymorphisms [ C825T, C1429T and G(-350)A] of the gene encoding the G protein beta 3 subunit (GNB3) and hypertension by performing a case-control study in the northern Han Chinese population. METHODS: We recruited 731 hypertensive patients and 673 control subjects (the calculated power value was > 0.8). Genotyping was performed to identify C825T, C1429T and G(-350)A polymorphisms using the TaqMan assay. Comparisons of allelic and genotypic frequencies between cases and controls were made by using the chi-square test. Logistic regression analyses were performed to investigate the relationships between the three polymorphisms of GNB3 gene under different genetic models (additive, dominant and recessive models). RESULTS: The genotype distribution and allele frequencies of C825T, C1429T and G(-350)A polymorphisms did not differ significantly between hypertensive patients and control subjects, either when the full sample was assessed, or when the sample was stratified by gender. No significant association was observed between C825T, C1429T and G(-350)A polymorphisms and the risk of essential hypertension in any genetic model. Linkage disequilibrium was only detected between C825T and C1429T polymorphisms. Haplotype analyses observed that none of the three estimated haplotypes significantly increased the risk of hypertension. CONCLUSIONS: Our study suggested that the GNB3 gene polymorphisms [C825T, C1429T and G(-350)A] were not significantly associated with essential hypertension in northern Han Chinese population.

Characterization of biochemical properties of Bacillus subtilis RecQ helicase.

RecQ family helicases function as safeguards of the genome. Unlike Escherichia coli, the Gram-positive Bacillus subtilis bacterium possesses two RecQ-like homologues, RecQ[Bs] and RecS, which are required for the repair of DNA double-strand breaks. RecQ[Bs] also binds to the forked DNA to ensure a smooth progression of the cell cycle. Here we present the first biochemical analysis of recombinant RecQ[Bs]. RecQ[Bs] binds weakly to single-stranded DNA (ssDNA) and blunt-ended double-stranded DNA (dsDNA) but strongly to forked dsDNA. The protein exhibits a DNA-stimulated ATPase activity and ATP- and Mg(2+)-dependent DNA helicase activity with a 3' → 5' polarity. Molecular modeling shows that RecQ[Bs] shares high sequence and structure similarity with E. coli RecQ. Surprisingly, RecQ[Bs] resembles the truncated Saccharomyces cerevisiae Sgs1 and human RecQ helicases more than RecQ[Ec] with regard to its enzymatic activities. Specifically, RecQ[Bs] unwinds forked dsDNA and DNA duplexes with a 3'-overhang but is inactive on blunt-ended dsDNA and 5'-overhung duplexes. Interestingly, RecQ[Bs] unwinds blunt-ended DNA with structural features, including nicks, gaps, 5'-flaps, Kappa joints, synthetic replication forks, and Holliday junctions. We discuss these findings in the context of RecQ[Bs]'s possible functions in preserving genomic stability.

[Associations between G1165C and A145G polymorphism of ß1-adrenoceptor and resting heart rate in the Northern Han Chinese].

OBJECTIVE: To systematically investigate the possible associations between G1165C and A145G polymorphism of ß1-adrenoceptor (ADRB1) and resting heart rate (HRrest) in Northern Han Chinese. METHODS: HRrest of 700 healthy Northern Han Chinese were measured in the sitting position.SNPs were genotyped by the TaqMan assay.Genotypes were differentiated by analyzing the fluorescence levels of PCR products using an ABI Prism 7900HT Sequence Detector. RESULTS: HRrest was significantly lower in A145G AA carriers than in AG and GG carriers (all P < 0.01) . Multiple linear regression analysis showed that age, smoking habits, systolic blood pressure, triglyceride, serum creatinine and A145G polymorphism were associated with HRrest (P < 0.01) . A145G was significantly related with HRrest independent of other possible confounding variables, and the partial regression coefficient was 2.148 (P < 0.05) . After adjusting for other confounding factors, significant association between A145G and HRrest was only found in male subjects (P < 0.05) but not in female subjects (P > 0.05) . CONCLUSION: The A145G polymorphism of ADRB1 gene is associated with HRrest in Northern male Han Chinese.

[Association of C602A and T1559C polymorphisms of E-selectin gene and essential hypertension].

OBJECTIVE: To investigate the possible genetic associations between the C602A and T1559C polymorphisms of E-selectin (SELE) and essential hypertension. METHODS: Essential hypertensive patients (n = 500) and healthy normotensive subjects (n = 930) were screened for the genotypes C602A and T1559C by real-time quantitative polymerase chain reaction after DNA extraction to identify representative variations in the SELE gene. RESULTS: Normotensive subjects and hypertensive patients were significantly different with respect to the genotypes CC, CA and AA, 26 (5.2%), 20 (4.0%) and 454 (90.8%) vs 14 (1.5%), 53 (5.7%) and 863 (92.8%) respectively of C602A. And the C-allele frequency was also significantly different between the NT and EH groups (C, A = 7.2%, 92.8% vs 4.4%, 95.6%). When subgrouped by gender, frequency of CC, CA, AA between normotensive and essential hypertensive males was 14 (4.7%), 11 (3.7%), 272 (91.6%) and 10 (1.7%), 34 (5.8%), 545 (92.5%), which differed significantly (P < 0.05), while in female groups, all the frequency of genotypes were significantly different (P < 0.01) except CC + CA. The additive model (TT, TC, CC) of the T1559C genotype was significantly different between essential hypertensive and normotensive groups overall, 57 (11.4%), 200 (40.0%), 43 (48.6%) and 66 (7.1%), 354 (38.1%), 510 (54.8%), respectively. The T-allele of hypertensive patients significantly differed from normotensive subjects (T, C = 31.4%, 68.6% vs 26.1%, 73.9% respectively). When subgrouped by gender, between the male NT and EH groups, the TT, TC and CC frequency of T1559C were 36 (5.9%), 117 (39.4%), 144 (48.5%) and 35 (5.9%), 230 (39.0%), 354 (55.0%), and the frequency of T vs C was 31.4% vs 68.6% and 26.1% vs 73.9%, which were significantly different (all P < 0.01). As in female NT and EH groups, there were not significant differences existed at all. CONCLUSION: C602A and T1559C of SELE are associated with essential hypertension in the Chinese population, and T1559C is closely related with male hypertension other than in females.

[Plasma proteomic analysis in male normotensive, prehypertensive and hypertensive subjects].

OBJECTIVE: To compare the plasma proteome among male normotensive, prehypertensive, and hypertensive subjects. METHODS: Plasma proteome was analyzed by two-dimensional electrophoresis combined with MALDI-TOF mass spectrometry in this case-control study among well matched male normotensive, prehypertensive and hypertensive subjects (n = 26 each). RESULTS: The results showed that there were 22 differentially expressed protein spots among the protein samples derived from the 3 groups which corresponded to 18 proteins associated with inflammation and immunity, lipid metabolism, transport, coagulation and fibrinolysis, cell proliferation and apoptosis, and antioxidation. CONCLUSION: Proteins were differentially expressed in male subjects with various blood pressure levels.

[Near-infrared reflectance spectroscopy analytic models established for the crude protein and crude fiber of alfalfa property].

Near-infrared reflectance spectroscopy (NIRS) calibrations of crude protein and crude fiber in 152 alfalfa samples were developed by means of partial least-squares (PLS) regression. Different interval wavelengths of 1, 2 and 5 nm and two kinds of samples (crude and fine) were set to collect spectral data and establish calibration respectively. The accuracy and repeatability of calibrations were verified so as to compare superiority and inferiority. Results showed that the best validation result is the calibration by 2 nm interval wavelength and fine sample, and the correlation coefficients of calibration (R2) were 0.97 and 0.94 for crude protein and crude fiber, and the SECV of these parameters were 0.42 and 0.78 respectively. The coefficients of determination for validation (R2(val)) were 0.96 and 0.92, and the SEP of these parameters were 0.43 and 0.79. The experiment showed that it is feasible to evaluate the major quality traits of alfalfa by NIRS. All together, it provided a new model to verify the crude protein and fiber compositions of alfalfa.

[Pericardial endothelin secretion in patients underwent open heart surgery.].

OBJECTIVE: To evaluate pericardial endothelin (ET) secretion by the human pericardial mesothelial cells. METHODS: Plasma, pericardial fluid and pericardial tissue were obtained in 51 patients receiving open heart surgery (coronary artery bypass grafting, elective heart valvuloplasty or valve replacement). ET concentrations in the plasma, pericardial fluid and pericardial tissues were measured by radioimmunoassay (RIA). ET mRNA expression in the human pericardium was detected by in situ hybridization. RESULTS: (1) The levels of ET in human pericardial fluid was significantly higher than that in the plasma [(128.8 +/- 44.0) ng/L vs. (93.7 +/- 28.6) ng/L, P < 0.001]; (2) ET concentration in the pericardial tissue was (510.3 +/- 156.7) ng/kg; (3) In situ hybridization technique evidenced the abundant ET mRNA expression in human pericardial mesothelial cells. CONCLUSION: The study indicated that pericardium secreted ET into the pericardial space.

[Study on the antimutagenicity of Cordyceps taii polysaccharide].

OBJECTIVE: To study the antimutagenicity of Cordyceps taii polysaccharide (CDPl). METHODS: Ames test and micronucleus form assay of polychromatic erythrocytes in mice bone marrow were used to evaluate the antimutagenic effect of CDPI with different concentrations. RESULTS: CDP1 could inhibit the reverse mutation on Salmonella typhimuriun strains TA97, TA98, TA100 and TA102 without S9 in different degree, especially remarkable in the strains T97 ,T98 and T102. The maxium inhibition rates of strains TA97, TA98, TA100 and T102 were 68.5%, 79.5%,19. 8% and 81.3%, respectively. In addition, CDPI could significantly reduce micronucleus frequencies induced by cyclophosphamide, and the rate of maxium inhibition was 70.3%. CONCLUSION: Cordyceps taii polysaccharide possesses significantly antimutagenic properties.

The zinc-binding motif of human RECQ5beta suppresses the intrinsic strand-annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme.

RecQ family helicases, functioning as caretakers of genomic integrity, contain a zinc-binding motif which is highly conserved among these helicases, but does not have a substantial structural similarity with any other known zinc-finger folds. In the present study, we show that a truncated variant of the human RECQ5beta helicase comprised of the conserved helicase domain only, a splice variant named RECQ5alpha, possesses neither ATPase nor DNA-unwinding activities, but surprisingly displays a strong strand-annealing activity. In contrast, fragments of RECQ5beta including the intact zinc-binding motif, which is located immediately downstream of the helicase domain, exhibit much reduced strand-annealing activity but are proficient in DNA unwinding. Quantitative measurements indicate that the regulatory role of the zinc-binding motif is achieved by enhancing the DNA-binding affinity of the enzyme. The novel intramolecular modulation of RECQ5beta catalytic activity mediated by the zinc-binding motif may represent a universal regulation mode for all RecQ family helicases.

[Increased plasma vasoactive substances and antioxidant enzymes levels in prehypertensive patients].

OBJECTIVE: This study investigates the plasma vasoactive substances and antioxidant enzymes levels in prehypertensive patients. METHODS: Patients were scruited according to JNC-7 and divided into three groups: 74 normotensive subjects (NT group, 38 males, mean age 47.15 +/- 7.77 years old); 51 prehypertensive patients (PH group, 29 males, mean age 47.82 +/- 5.16 years old) and 71 essential hypertensive patients (EH group, 37 males, mean age 48.25 +/- 7.97 years old). Serum lipids and plasma angiotensin II (Ang II), endothelin (ET), vasopressin (AVP), calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), superoxide dismutase (SOD) and glutathione peroxidase (GPX) by radioimmunoassay and enzyme linked immunosorbent assay. RESULTS: Serum Lipids (TG, CHO and LDL) were significantly higher in the PH and EH groups compared to NT group (all P < 0.05). Ang II, AVP and ET were significantly increased while CGRP decreased in the EH group than that in NT group (all P < 0.05). SOD was significantly lower while GPX significantly higher. Further more, in the PH and EH groups than those in the NT group (all P < 0.05). CONCLUSION: SOD was reduced and GPX increased in prehypertensive patients.

Human vascular smooth muscle cells and endothelial cells cocultured on polyglycolic acid (70/30) scaffold in tissue engineered vascular graft.

BACKGROUND: Current prosthetic, small diameter vascular grafts showing poor long term patency rates have led to the pursuit of other biological materials. Biomaterials that successfully integrate into surrounding tissue should match not only the mechanical properties of tissues, but also topography. Polyglycolic acid (70/30) has been used as synthetic grafts to determine whether human vascular smooth muscle cells and endothelial cells attach, survive and secrete endothelin and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha). METHODS: Endothelial cells and smooth muscle cells were isolated from adult human great saphenous vein. They were seeded on polyglycolic acid scaffold in vitro separately to grow vascular patch (Groups A and B respectively) and cocultured in vitro to grow into vascular patch (Group C). Smooth muscle cells and endothelial cells were identified by immunohistochemical analysis and growth of cells on polyglycolic acid was investigated using scanning electron microscopy. The levels of endothelin and 6-keto-PGF1alpha in the culturing solutions were examined by radioimmunology to measure endothelial function. RESULTS: Seed smooth muscle cells adhered to polyglycolic acid scaffold and over 28 days grew in the interstices to form a uniform cell distribution throughout the scaffold. Then seed endothelial cells formed a complete endothelial layer on the smooth muscle cells. The levels of endothelin and 6-keto-prostaglandin F1 alpha in the culturing solution were (234 +/- 29) pg/ml and (428 +/- 98) pg/ml respectively in Group C and (196 +/- 30) pg/ml and (346 +/- 120) pg/ml in Group B; both significantly higher than in Groups A and D (blank control group, all P < 0.05). CONCLUSIONS: Cells could be grown successfully on polyglycolic acid and retain functions of secretion. Our next step is to use human saphenous vein smooth muscle cells and endothelial cells to grow tubular vascular grafts in vitro.

[Hyperplasia suppressor gene expression in vascular smooth muscle cells derived from normotensive and hypertensive patients underwent bypass surgery].

OBJECTIVE: To investigate the novel hyperplasia suppressor gene (HSG) expression in vascular smooth muscle cells derived from normotensive and hypertensive patients underwent bypass surgery. METHODS: Coronary heart disease patients underwent coronary artery bypass graft (CABG) operation in BEIJING ANZHEN hospital from 4 - 9, 2006 were enrolled in this study and divided into hypertensive group (n = 28) and normotensive group (n = 26). The preoperative venous blood samples were taken for serum biochemical and vasoactive peptides measurements. Total RNA was extracted from WBC, explanted-vessels and cultured VSMCs using TRIZOL and HSG expression was determined by Semi-Quantitative RT-PCR. RESULTS: Body mass index (BMI) was significantly higher in hypertensive group compared to normotensive group (P < 0.01) while other biochemic parameters and vasoactive peptides were similar between the groups. BMI and GLU, BMI and SBP, BMI and DBP, GLU and TG, SBP and DBP were positively correlated (all P < 0.05). HSG expression in WBC, VSMCs and vessel tissue were significantly lower in hypertensive group than those in normotensive group (all P < 0.05). HSG expression in tissue was negatively correlated to BMI, SBP and DBP (all P < 0.05). CONCLUSIONS: Reduced HSG expression and the negative correlation on vascular tissue HSG expression to BMI, SBP and DBP suggested a possible inhibitory role of HSG on VSMC proliferation and blood pressure.

[Blood vessel tissue engineering: seeding vascular smooth muscle cells and endothelial cells sequentially on biodegradable scaffold in vitro].

OBJECTIVE: To evaluate the effect of seeding vascular smooth muscle cells and endothelial cells sequentially on biodegradable scaffold in vitro. METHODS: Venous endothelial cells and smooth muscle cells of artery were isolated from the umbilical cord of newborn and cultured in vitro. The cultured arterial smooth muscle cells and cultured endothelial cells were seeded on the polyglycolic acid and polylactide (PLGA) nonwoven mesh as tissue scaffolds respectively (group A and group B). And cultured endothelial cells were seeded on the scaffold already seeded with smooth muscle cells sequentially to construct vascular patch (group C). One month after cultivation the patches of the 3 groups underwent HE staining and microscopic examination and scanning electron microscopy. Immunohistochemistry was used to examine the existence of actin and factor VIII-related antigen, secreted by endothelial cells, in the cells. The levels of endothelin and 6-Keto-PGF1alpha in the culture solution were examined by means of radioimmunology so as to measure the endothelial function. Pure culture solution was used as blank control (group D). RESULTS: Microscopy showed that cells were fused into patch in the group C. HE staining showed that the smooth muscle cells were embedded into the PLGA scaffold and grew in multiple layers covered by a layer of endothelial cells on the surface. The types of cells could be identified by immunohistochemical procedure. The levels of endothelin and 6-Keto-PGF1alpha in the culture solution were 229 pg/ml +/- 34 pg/ml and 402 pg/ml +/- 108 pg/ml respectively in the group C, and 200 pg/ml +/- 31 pg/ml and 336 pg/ml +/- 121 pg/ml in the group B, both significantly higher than those in the groups A and D (blank control group, all P < 0.05 or P < 0.05). CONCLUSION: Tissue engineering blood vessel with normal morphological and functional characters to a certain degree can be constructed through seeding cultured vascular smooth muscle cells and endothelial cells sequentially on PLGA scaffold.

[Relationship of the HSG gene single nucleotide polymorphism and hypertension].

OBJECTIVE: To investigate the single nucleotide polymorphism (SNP) of the novel hyperplasia suppressor gene (HSG) to uncover the relationship between HSG SNP and hypertension. METHODS: Totally, 74 normotensive people (38 men and 36 women), 51 patients with essential hypertension (27 men and 24 women) and 20 hypertensive patients with family history of essential hypertension (9 men and 11 women) were chosen, with mean ages of (54 +/- 8) years, (57 +/- 8) years and (38 +/- 22) years, respectively. Peripheral venous blood specimen was collected from each of them and then DNA was extracted. The right primers were designed for DNA amplification with PCR. Each of the PCR-products from different groups was sequenced by ABI PRISM 377-DNA sequencer and their base components and characteristics of the same fragment were compared each other. RESULTS: Blood levels of creatinine (CRE) and urea nitrogen (BUN) were significantly higher in the hypertensives than in the normotensives (P < 0.01). Systolic and diastolic blood pressures were significantly higher in the hypertensives and the hypertensives with family history of essential hypertension than in the normotensives (P < 0.01 and P < 0.05). There existed three kinds of SNP in the HSG 12th intron (1q82139 G/A, 82153C/G and 82273G/-), and there was significant difference in 1q82153C/G and 82273G/- SNP between the hypertensives and normotensives (P < 0.05 and P < 0.01) and between the hypertensives with family history and the normotensives (P < 0.01 and P < 0.01). And, the similar difference in C/G allele and G deletion could be found in different populations. Moreover, the CC genetype of 1q82153 was common in the population (P < 0.01) and G deletion was more common in Chinese Han people with family history of essential hypertension. There was no significant difference existed in 1q82139 G/A mutation among the three groups. CONCLUSIONS: Measurements of renal function indicators (CRE and BUN) could probably reflect earlier advance of hypertension and damage to target organs. There existed three kinds of mutation in the 12th intron of the HSG 1q82139 G/A, 82153 C/G and 82273 G/-. The 1q82139 G/A could be a nonsense mutation and there was significant difference in the 1q82153 C/G and 1q82273 G/- SNP and gene frequencies between different Chinese Han populations, which could be independent risk factors for essential hypertension.

The zinc finger motif of Escherichia coli RecQ is implicated in both DNA binding and protein folding.

The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys397 and Cys400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.