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Pu’er Tea is a popular post-fermented tea and has a variety of health benefits. Many microorganisms, especially lactobacillus, exist and play an important role in the fermentation process of Pu’er tea. In this study, we try to screen some lactobacillus by plate method from the Pu’er tea, and strain C2 with significant cycle isolated from Pu’er tea. 16S rRNA gene sequencing results showed that strain C2 had 99 % sequence similarity to that of the type strain of three Bacillus coagulans (NR 102791.1, CP002472.1, NR 115727.1), and the results show that methyl red test and V&P test were positive. Sugar fermentation test suggested that strain C2 produced acid but not gas, which is identical to B. coagulans. This study confirmed that lactic acid bacteria play a role in Pu’er tea fermentation.

Human epidermal growth factor (hEGF), a well-known polypeptide agent which has been widely used in the medicine and cosmetics, is a 6.2 kDa single-chain polypeptide consisting of 53 amino acids. Here, to enhance its transmembrane ability, a recombinant EGF fused with Pep-1, a cell-penetrating peptides (CPP) that has been previously shown to be powerful transport vector tool for the intracellular delivery of a variety of cargos through the cell membrane, was expressed in Escherichia coli. Furthermore, The expression conditions was optimized, and the results showed that the Pep-1-fused EGF (P-EGF) could be successfully expressed in E. coli BL21-TrixB (DE3) using an expression vector, pGEX-6P-3, which contains a GST tag. The recombinant product reached the highest soluble expression when the expression strain was induced by 0.2 mmol/l IPTG and cultivated at the temperature of 20 °C with a rotation speed of 200 rpm for 8 h.

In response to stress signals, postnatal cardiomyocytes undergo hypertrophic growth accompanied by activation of a fetal gene program, assembly of sarcomeres, and cellular enlargement. We show that hypertrophic signals stimulate the expression and transcriptional activity of myocardin, a cardiac and smooth muscle–specific coactivator of serum response factor (SRF). Consistent with a role for myocardin as a transducer of hypertrophic signals, forced expression of myocardin in cardiomyocytes is sufficient to substitute for hypertrophic signals and induce cardiomyocyte hypertrophy and the fetal cardiac gene program. Conversely, a dominant-negative mutant form of myocardin, which retains the ability to associate with SRF but is defective in transcriptional activation, blocks cardiomyocyte hypertrophy induced by hypertrophic agonists such as phenylephrine and leukemia inhibitory factor. Myocardin-dependent hypertrophy can also be partially repressed by histone deacetylase 5, a transcriptional repressor of myocardin. These findings identify myocardin as a nuclear effector of hypertrophic signaling pathways that couples stress signals to a transcriptional program for postnatal cardiac growth and remodeling.

Telomeres are critical in maintaining chromosome and genomic stability. Arsenic, a human carcinogen as well as an anticancer agent, is known for its clastogenicity. To better understand molecular mechanisms of arsenic actions, we investigated arsenite effects on telomere and telomerase and determined cell growth and apoptosis in HL-60 and HaCaT cells in vitro. Low concentrations (0.1-1 microM in HaCaT and 0.1-0.5 microM in HL-60) of arsenite increased telomerase activity, maintained or elongated telomere length, and promoted cellular proliferation. High concentrations (>1-40 microM) of arsenite decreased telomerase activity, telomere length and induced apoptosis. Results from the studies comparing cell lines with and without telomerase activity suggested that telomerase was involved in arsenic-induced apoptosis. The spin trap agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was effective in protecting the arsenite-induced telomere attrition and apoptosis, suggesting that reactive oxygen species may play an important role in the shortening of telomeres and apoptosis induced by arsenic. These findings suggest the carcinogenic effects of arsenic may be partly attributed to increase in telomerase activity leading to promotion of cell proliferation and its anticancer effects by exerting oxidative stress and leading to telomeric DNA attrition and apoptosis.

Opposite biological effects of arsenic trioxide (As2O3) and arsacetin on the growth of human gastric cancer MGC-803 cells have been observed. Results show that As2O3 inhibited the growth of MGC-803 cells by triggering apoptosis, whereas arsacetin promoted the cell proliferation and seemed to stimulate the secretion of some growth factors at the same micromolar concentrations. Further studies showed that As2O3 could regulate protein tyrosine kinase activity, protein tyrosine phosphorylation, and Bcl-2 protein and upregulate p53 protein. The ability of arsacetin to promote cell proliferation is linked with causing the opposite effects on these factors. These results indicate that the opposite biological effects of As2O3 and arsacetin involve different regulations of molecular mechanisms in MGC-803 cells and that arsacetin may be a potential tumor promoter.

Connexin 43 (Cx43), the principal gap junction protein in vascular smooth muscle cells(VSMCs) resulting in modulation of gap junction. Myocardin is thought to have a key role in Vascular Smooth Muscle Cell (VSMC) development by acting on CArG-dependent genes. In this study, a human Cx43 promoter luciferase reporter construct was generated by PCR amplification of Cx43 promoter. The PCR fragment was digested and cloned into pGL3-Basic vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of Cx43 promoter were successfully constructed. Then the effects of a key transcription factor, which plays important roles in Cx43 levels control, were investigated by luciferase reporter assays in HEK293. The results showed that myocardin can enhance transcriptional activity of Cx43. Our observations provide evidence that Myocardin targets Cx43 expression directly.

Vascular endothelial cadherin (VE-cadherin) is the major determinant of endothelial cell contact integrity and is required in vascular development and angiogenesis. Serum response factor (SRF) plays essential roles in postnatal retinal angiogenesis and adult neovascularization. It is unclear whether transcription of VE-cadherin is mediated by a SRF co-activator, myocardin-related transcription factor-A (MRTF-A). Here we have demonstrated that MRTF-A is a key regulatory factor to activate the transcription of VE-cadherin in human umbilical vein endothelial cells (HUVECs). siRNA-mediated knockdown of MRTF-A decreased the level of VE-cadherin in HUVECs. Vascular endothelial growth factor (VEGF) induced MRTF-A binding to the SRF-binding site (CArG box) within VE-cadherin promoter. Histone acetyltransferase p300 and MRTF-A could synergistically augment the expression of VE-cadherin by enhancing acetylation of histone3K9 (H3K9Ac), histone3K14 (H3K14Ac) and histone4 at the SRF-binding site within VE-cadherin promoter. Taken together, these data identified a detailed regulatory mechanism of VE-cadherin gene expression.

The JAK-STAT3 signaling pathway is one of the critical pathways regulating cell proliferation, differentiation, and apoptosis. Myocardin is regarded as a key mediator for the change of smooth muscle phenotypes. However, the relationship between STAT3 and myocardin in the vascular smooth muscle cell (VSMC) phenotypic switch has not been investigated. The goal of this study was to investigate the molecular mechanism by which STAT3 affects the myocardin-regulated VSMC phenotypic switch. Data presented in this study demonstrated that STAT3 was rapidly up-regulated after stimulation with VEGF. Inhibition of the STAT3 activation process impaired VSMC proliferation and enhanced the expression of VSMC contractile genes by increasing serum-response factor binding to the CArG-containing regions of VSMC-specific contractile genes. In contrast, the interaction between serum-response factor and its co-activator myocardin was reduced by overexpression of STAT3. In addition, treated VEGF inhibited the transcription activity of myocardin, and overexpression of STAT3 inhibited myocardin-induced up-regulation of VSMC contractile phenotype-specific genes. Although myocardin and STAT3 are negatively correlated, interestingly, both of them can enhance the expression of VEGF, suggesting a feedback loop to regulate the VSMC phenotypic switch. Taken together, these results indicate that the JAK-STAT3 signaling pathway plays a key role in controlling the phenotypic switch of VSMCs through the interactions between STAT3 and myocardin by various coordinated gene regulation pathways and feedback loops.

Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate β cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes.

In this paper, a heart-cutting two-dimensional high-performance liquid chromatography coupled with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was established for controlling the quality of different batches of Hypericum ascyron extract for the first time. In comparison with the common one-dimensional fingerprint, the second-dimensional fingerprint compiled additional spectral data and was hence more informative. The quality of H. ascyron extract was further evaluated by similarity measures and the same results were achieved, the correlation coefficients of the similarity of ten batches of H. ascyron extract were ＞0.99. Furthermore, we also evaluated the quality of the ten batches of H. ascyron extract by antibacterial activity. The result demonstrated that the quality of the ten batches of H. ascyron extract was not significantly different by MTT. Finally, we demonstrated that the second-dimensional fingerprint coupled with the MTT method was a more powerful tool to characterize the quality of samples of batch to batch. Therefore the proposed method could be used to comprehensively conduct the quality control of traditional Chinese medicines.

According to previous studies, hyperoside possesses myocardial protective effects. To investigate whether isoquercitrin and isohyperoside have similar functions, the protective effects of isoquercitrin and isohyperoside against H2O2‑induced injury in H9c2 rat cardiomyoblasts were evaluated using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay. The mechanism of action was investigated by assessing the leakage of lactate dehydrogenase (LDH) of hyperoside and isoquercitrin‑pretreated H9c2 cardiomyocytes following H2O2‑induced injury, and examining their effects on the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) activity. The isolation of two flavonoid glycosides from H. ascyron was performed, following extraction, using semi‑preparative high performance liquid chromatography. Using the spectral characteristics, the structures of these compounds were identified as isoquercitrin and isohyperoside. This was the first time, to the best of our knowledge, that isohyperoside has been identified from H. ascyron. The results revealed that isoquercitrin and isohyperoside possessed similar protective effects to hyperoside against H2O2‑induced injury in H9c2 cells. The half maximal inhibitory concentration values of hyperoside, isoquercitrin and isohyperoside were 0.0008, 0.0017 and 0.0002 µM, respectively. Based on these results, isohyperoside possessed more marked protective effects against H2O2‑induced injury in the H9c2 cardiomyoblasts. The significant reduction in LDH leakage, decrease in MDA level and increase in SOD activity also provided evidence of the cardioprotective effects of isoquercitrin and isohyperoside. The present study reported for the first time, to the best of our knowledge, the myocardial protective effects of isoquercitrin and isohyperoside. The mechanism of action may involve protection of the cell membrane from oxidative damage.

High risk human papillomavirus (HPV) is a well recognized causative agent of cervical cancer. Suberoylanilide hydroxamic acid (SAHA) is a potential anti-cervical cancer drug; however, its effect on the expression of HPV E6 and E7 genes remains unclear. Here, we show that, in SAHA treated HeLa cells, HPV18 E6 and E7 mRNA and protein levels were reduced, HPV18 promoter activity was decreased, and the association of RNP II with HPV18 promoter was diminished, suggesting that SAHA inhibited the transcription initiation of HPV18 E6 and E7 genes. In SAHA-treated HeLa, although the level of lysine 9-acetylated histone H3 in the whole cell extracts increased obviously, its enrichment on HPV18 promoter was significantly reduced which is correlated with the down-regulation of HPV E6 and E7.

Human chorionic gonadotropin (hCG) is a glycoprotein produced by placental trophoblasts. Previous studies indicated that hCG could be responsible for the pregnancy-induced protection against breast cancer in women. It is reported that hCG decreases proliferation and invasion of breast cancer MCF-7 cells. Our research also demonstrates that hCG can reduce the proliferation of MCF-7 cells by downregulating the expression of proliferation markers, proliferating cell nuclear antigen (PCNA), and proliferation-related Ki-67 antigen (Ki-67). Interestingly, we find here that hCG elevates the state of cellular differentiation, as characterized by the upregulation of differentiation markers, β-casein, cytokeratin-18 (CK-18), and E-cadherin. Inhibition of hCG secretion or luteinizing hormone/hCG receptors (LH/hCGRs) synthesis can weaken the effect of hCG on the induction of cell differentiation. Furthermore, hCG can suppress the expression of estrogen receptor alpha. hCG activated receptor-mediated cyclic adenosine monophosphate/protein kinase A signaling pathway. These findings indicated that a protective effect of hCG against breast cancer may be associated with its growth inhibitory and differentiation induction function in breast cancer cells.

Breast cancer is the leading cause of cancer death in women worldwide. It is well known that oncogene activation and anti-oncogene inactivation affect the development and progression of breast cancer, but the role of oncogene activation and anti-oncogene inactivation in breast cancer is still not fully understood. We now report that maspin acts as a tumor suppressor gene to induce MCF-7 cell apoptosis. In addition, maspin promoter hypermethylation and histone hypoacetylation lead to silencing of maspin gene expression in MCF-7 cells. Moreover, DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dc) and/or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) strongly up-regulated the expression of maspin in MCF-7 cells. Notably, myocardin can promote the re-expression of maspin in MCF-7 cells. Luciferase assay shows that myocardin activates the transcription of maspin promoter by CArG box. More importantly, 5-aza-dc/TSA and myocardin synergetically enhance re-expression of maspin and augment maspin-mediated apoptosis in MCF-7 cells. Thus, these data reveal the new insight that myocardin meditates apoptosis in breast cancer through affecting maspin re-expression and epigenetic modification to regulate the development of breast cancer, thereby raising the possibility of its use in breast cancer therapy.

Arsenic trioxide (As2O3), used to treat human diseases for centuries in traditional Chinese medicine, has been identified as a very effective antileukaemic agent, but its effect on solid tumours which could be more suitable for clinical treatment with arsenic compounds is still unknown. In this study, we investigated the in vitro effect of As2O3 at concentrations of 0.01-1 microM against six human malignant cell lines, MGC-803, HIC, MCF-7, HeLa, BEL-7402 and A549 cells. As2O3 inhibited growth and induced apoptosis in these malignant cells at varying degrees, in a time dose-dependent manner. The most marked effects were seen in the gastric cancer cell line, MGC-803. In contrast, minimal growth inhibition and induction of apoptosis occurred in human embryonic pulmonary cells following treatment with As2O3 found at the same concentrations. Changes in intracellular Ca2+, following As2O3 treatment were measured by Ca2+ sensitive fluorescent probe Indo-1/AM in flow cytometric assays. The increase in intracellular Ca2+ correlated with the sensitivity of these cells to As2O3, possibly indicating that a critical intracellular Ca2+ signal transduction pathway could be involved in As2O3-mediated cell-death and its selectivity. The marked sensitivity of MGC-803 cells in vitro suggests that As2O3 may be a potential antigastric cancer agent.

Lycopene, as a red-colored intermediate of the β-carotene biosynthetic pathway, has attracted considerable attention due to its beneficial role in human health. And it is a potent antioxidant and of much bioactivity such as quenching singlet oxygen and scavenging free radicals, resulting in protection against oxidative DNA damage in vitro and in vivo. In this review we hope to concisely describe the nature of lycopene compounds, the scientific basis for its proposed anti cancer and cardiovascular protection properties and focus on discussion of such advances in microbial production of lycopene by fermentation.

Cancer is one of the major causes of death worldwide. Many clinical evidences showed that the incidence of colon cancer increased after the cholecystectomy, suggesting that bile acid and its derivatives might play roles in the process of tumorigenesis and its prevention. Bile acid derivative is a kind of good candidates for prodrug design, but there are still few reports of its development for anticancer drugs either domestically or internationally. In the present study, IC50 of 9 bile acid derivatives against MCF-7 human breast cancer cells was detected by MTT assay, and the safety of several bile acid derivatives with high anticancer effect were further analyzed in human umbilical vein endothelial cells. The results showed that among 9 bile acid derivatives, cholic acid n-buthyl ester had lower IC50 against cancer cells, but it did not have cytotoxicity to normal cell at low concentration, indicating that they would be a good candidate for the development of novel anticancer agents.

In this paper, we focus on the effects of dual-frequency ultrasound (40 and 80 kHz) with α-amylases (5U/g) on the properties and structure of mung bean starch. The reducing sugar value and starch solubility in the treated starch slurry under ultrasound enzyme were studied. Scanning electron microscopy (SEM) and Fourier transform infrared spectrometer (FTIR) were used to measure the apparent structure and crystal texture of starch. The results indicated that compared with the results of single-frequency ultrasound treatment, dual-frequency ultrasound promoted the starch hydrolysis by amylase and increased the starch solubility remarkably. More channels and holes were found on the surface of treated starch granule by SEM. The results of FTIR analysis showed the FTIR peak shape of ultrasonic treated starch was different from that of the control starch.

MicroRNAs (miRNAs) are small noncoding RNAs with regulatory functions, which play an important role in malignancies. An increasing amount of experimental evidence has shown that many miRNAs are aberrantly expressed in breast cancer and influence breast cancer behavior and progression. Furthermore, miRNAs can act either as tumor suppressors or as oncogenes, depending on the targets they regulate, and measurements of miRNAs expression in breast cancer have diagnostic and prognostic implications. Thus, this implies that miRNAs have huge potential as biomarkers. In addition, their extreme stability and ease of detection further support the idea that miRNAs have great potential to evolve into effective biomarkers in the clinic. The objective of this review is to update current realization regarding that miRNAs are promising candidates at diagnostic, therapeutic and prognostic evaluation aspects of clinical application.

Breast cancer, a hormone-dependence disease, is the most common malignant tumor in women. Breast cancer incidence rates increase with age. Telomerase is a ribonucleoprotein that maintains chromosome ends and endows cells with unlimited proliferative potential. Activation of telomerase maintains a relatively stable telomere length, confers immortality on some cells, and may even lead to cancer. It has been reported that, in the tissues of the breast, the activity of telomerase can be detected in more than 90 % of invasive breast cancers, while not in adjacent normal tissues. Thus, telomerase activation plays a critical role in development of breast cancer. Telomerase has been proposed as a biomarker with diagnostic and prognostic potential in breast cancer as well as a basis for the targeted therapy of breast cancer.

Myocardin-related transcription factor A (MRTF-A) is a transcriptional coactivator of serum response factor (SRF). Recent studies have found that the MRTF–SRF signal pathway might play critical role in Rho-dependent cytoskeletal rearrangements and tumor metastasis. To further explore the detailed molecular mechanisms for MRTF-A-mediated cell migration, in this study, the effect of MRTF-A on the migration of mouse fibroblast NIH3T3 cells and MCF-7 human breast cancer cells was detected using a scratch wound model, and its transcriptional activity on the promoter of Cysteine-rich angiogenic inducer 61 (CYR61) was also analyzed by luciferase assay. The results showed that overexpression of MRTF-A could significantly promote the migration of NIH3T3 cells and drive the transcriptional activity of CYR61 promoter in African green monkey kidney COS-7 cells and strongly enhance upregulation of CYR61 gene. In contrast, RNA interference-mediated knockdown of MRTF-A reduced the migration ability of MCF-7 breast cancer cells. Taken together, our results suggested that MRTF-A is involved in tumor metastasis via transactivation of CYR61.

Biological treatment is one of the considerable choices for removing of organic pollutants present in petrochemical wastewaters. In this study, five strains, named as BS1, BS2, BS3, BS4 and BS5, were isolated from sludge nearby a petroleum smelter. BS5, the isolate with the highest COD removal rate, was identified as Bacillus flexus, based on 16S rDNA sequences. Subsequently, the optimized COD removal conditions of BS5 were investigated. It was indicated that the optimal conditions were 0.5 % corn starch, 1 % corn steep liquor, 35 C, pH 7.5, and 10 % (v:v) inoculation size. Under such circumstance, the removal rate of COD can reach 81.04 %. The isolation of Bacillus flexus strain BS5 provided an alternative for the bioremediation of alkaline wastewater. Lastly, the study showed that consecutive disposal process may help to reducing COD of wastewater effectively.

Objective: Monitoring data of patients with essential hypertension associated with impaired glucose tolerance then provide clinical basis for the intervention of diabetes and reduce cardiovascular and cerebrovascular disease risk factors. Method: Select 158 patients without sugar metabolic abnormalities previously, whose fasting plasma glucose is

MicroRNAs are small non-coding RNAs with 19-22 nucleotides. MiR-124 expresses abundantly in brain tissue and is involved in the development of ischemic cerebrovascular disease (ICVD). Tumor necrosis factor-alpha (TNF-α) is one of the important factors of the inflammatory reaction after cerebral ischemia. To explore the roles of miR-124 and TNF-α in the process of ICVD, here, we collected the serum of 15 patients with ICVD and 5 normal individuals. The levels of miR-124 and TNF-α were detected by real-time PCR and ELISA. The results showed that compared with the normal individuals, the levels of miR-124 and TNF-α in serum were upregulated in the patients, while they were downregulated after clinical treatment, suggesting that these two factors could be used as good biomarkers for the diagnosis and prognosis of ICVD. Besides, our research also provided a theory foundation for understanding the mechanism of inflammatory response in cerebral ischemia mediated by miR-124.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease leading to synovial hyperplasia and a series of multisystem comorbidities. Permanent disability usually occurs in 20–30 % of untreated patients within 2–3 years of the disease. Therefore, it is important to diagnose the disease and initiate treatment as early as possible. The drug therapy plays an important role in maintaining a state of very low disease activity and slowing the progression of joint damage. Traditional drug therapy in RA is a pyramid approach, in which nonsteroidal anti-inflammatory drugs (NSAIDs) serve as the base of pyramid and disease-modifying antirheumatic drugs (DMARDs) are employed relatively late. But since this approach is no longer valid, many new drugs used to manage RA have been developed. In this review, we specifically summarize the current drug therapies for the treatment of RA and briefly describe the etiology, pathology and pathogenesis of RA.

Mesenchymal stem cells (MSCs) are pluripotent progenitors and can differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes. Vascular endothelial growth factor (VEGF) has been shown to stimulate endothelial cell mitogenesis and migrations. To investigate whether VEGF could induce rat bone marrow-derived MSCs differentiation into endothelial cells, we isolated and cultured rat bone marrow-derived MSCs from the femur of the Sprague–Dawley rats. MSCs were treated with VEGF for 2, 4, 7 days. The cellular morphology changes of MSCs were observed under phase contrast microscope. Then we tested the expression of endothelial cell markers VEGF receptor fms-related tyrosine kinase 1 (Flt-1) by reverse-transcription PCR (RT-PCR), western blot, and immunofluorescence technology. Our results showed that the expression levels of Flt-1 were significantly increased in time-dependent manner. The above results show that VEGF can induce mesenchymal stem cells differentiation into endothelial cells. The data provides the basis of constructing tissue engineering vascular.

This paper described a combined high-performance liquid chromatography (HPLC) -photochemical spectrofluorimetry/solid-phase extraction (SPE) procedure for determining the concentration of unmetabolized folic acid in human serum. After being extracted by SPE (C18-H) column, the serum samples were irradiated under the UV lamp for 11 h, separated on a C18 HPLC column with methanol/water as the mobile phases and determined by a fluorescence detector. Fluorescence excitation and emission wavelengths were at 280 and 443 nm, respectively. The linear range was 1.9 ~ 8.0 ng/mL. The recovery was from 88.9 to 123.6 % with RSD in the range of 4.1–6.4 %.

Gambogic acid (GA) shows important biological activities for carcinoma cells, such as selectively inducing apoptosis and differentiation, inhibiting proliferation and transference, inversing multiple drug resistance (MDR). However, its aqueous solubility is so low that its oral bioavailability is not as good as those marketed antitumor chemical drugs. In this paper, two GA derivatives with better aqueous solubility were designed and prepared. Among them N,N-bis-(β-hydroxyethyl) GA carboxamide was synthesized by DCC-HOBt catalyzed coupling reaction between GA and diethanolamine, and monoester of succinic acid by GA esterification with methyl succinyl chloride followed by mild hydrolysis with lithium hydroxide. The structures of these two target compounds and one intermediate were characterized by ESI–MS and 1H NMR spectra after their purification. These studies are of great significance for us to develop GA chemical entities with effective potency to treat cancers.

In a number of pathological conditions such as hypertension, valvular disease, myocardial infarction, and cardiomyopathy, postnatal cardiomyocytes undergo cardiac hypertrophy. Cardiac hypertrophy is the heart’s response to increased biomechanical stress, which is accompanied by activation of a fetal gene program, assembly of sarcomeres, and cellular enlargement. Prolonged hypertrophy is a major predictor of heart failure and sudden death. Identification of the signaling molecules is necessary for the development of therapeutic strategies to prevent or reverse the disease. Recent researches have found that there were different signaling pathways activated in cardiac hypertrophy, but the molecular mechanism of cardiac hypertrophy is not completely elucidated so far. In this review, we summarize recent insights into hypertrophic signaling pathways.

Betulin and betulinic acid are natural product with antitumor and anti-HIV-1 activity. Betulin can be got by chemical extraction from a lot of plants. Betulinic acid can be synthesized from betulin. In order to get high biological activity compounds with antitumor and anti-HIV-1 activity from the bark of the white birch, the extraction and purification method of betulin was studied; the optimized extraction and purification condition was obtained. The two-step synthetic routes of betulinic acid from betulin were discussed. Betulinic acid was synthesized by oxidizing betulin with Jones reagent, followed by selective reduction with NaBH4. IR, HPLC–MS, H1-NMR were used to identify the structure of betulin and betulinic acid.

Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.

Myocardin-related transcription factor-A (MRTF-A) is a Rho signal-responsive transcriptional coactivator of serum response factor (SRF). Recent studies indicated that MRTF-A might be an important regulator of mammary gland and be involved in cancer metastasis. However, the roles of histone modification in the MRTF-A-dependent signal pathway and tumor migration are still not very clear. Here, we report that histone methylation is required for the MRTF-A-mediated upregulation of myosin regulatory light chain 9 (MYL9), an important cytoskeletal component which is implicated in cell migration. Furthermore, we demonstrate that SET and MYND domain containing protein 3 (SMYD3), a hitone methyltransferase (HMT) associated with carcinogenesis, might be the one which is responsible for the histone methylation occurred in the MRTF-A-mediated- transactivation of MYL9 and migration of breast cancer cells. Overexpression of SMYD3 promotes MRTF-A-mediated upregulation of MYL9 and migration of MCF-7 breast cancer cells, while contrary results were observed when the endogenous MRTF-A and SMYD3 were suppressed with specific siRNAs. In addition, the mutation analysis suggested that this cooperative transactivation is mainly mediated via the proximal binding element of MRTF-A in the promoter of MYL9, and the HMT activity of SMYD3 is required as well. Our findings reveal a new mechanism by which MRTF-A and SMYD3 functions in transcriptional regulation and cell migration, and provide a better understanding for metastasis of breast cancer.

Previous studies have shown that lactic acid bacteria can inhibit inflammatory responses, but the mechanisms are very little known. In this study, transaction and expression of three proinflammatory factors, iNOS, PTGS-2, and IL8, which are closely related to the inflammatory response, were investigated by luciferase reporter assay and RTPCR in HT-29 cells treated by Lactobacillus acidophilus. The results showed that the live L. acidophilus sharply down-regulated the transcription of these three genes. Because there was a NF-κB binding site located at -265 bp, -225 bp, and -95 bp upstream of the iNOS, PTGS-2, and IL8 promoters, respectively, we further addressed the effects of NF-κB on transaction of the three promoters by cotransfection. As was expected, NF-κBs remarkably upregulated the activity of the reporter gene and, no effect of NF-κBs on IL-8 promoter transaction was found after NF-κB binding site mutation of the IL8 promoter in HT-29 cells. In conclusion, the live L. acidophilus decreased the transcriptional activity of NF-κB and, in turn, inhibited the transaction of NF-κB on the three proinflammatory factors mentioned above.

Mesenchymal stem cells (MSCs) are pluripotent progenitors that can differentiate into a variety of cell types. Vascular endothelial growth factor (VEGF) is one of the major factors of initiating and regulating angiogenesis. It has been reported that VEGF can induce MSCs differentiated into endothelial cells (ECs). However, the mechanism that VEGF-induced MSC differentiation is not completely understood. Here, we showed that VEGF induced human and rat bone marrow-derived MSCs differentiation to ECs. Rho family plays an important role in VEGF-induced endothelial cell migration and angiogenesis. Our results indicated that in MSCs, VEGF activated Rho/ROCK signaling pathway and promoted nuclear translocation of myocardin-related transcription factor-A (MRTF-A), which is controlled by Rho/ROCK signaling. In addition, Rho inhibitor C3 transferase, ROCK inhibitor Y27632 or depletion of endogenous MRTF-A abolished the VEGF-induced differentiation of MSCs into ECs. Furthermore, VEGF also enhanced the expression levels of CYR61/CCN1, as a regulator of vascular development and angiogenesis, and knockdown of endogenous MRTF-A reduced VEGF-induced the upregulation of CYR61/CCN1. Report assays with site-direct mutation analysis of CYR61/CCN1 promoter demonstrated that MRTF-A transactivated CYR61/CCN1 promoter mainly depending on CArG box. In this study, we identify the Rho/MRTF-A signaling pathway as a main actor in controlling VEGF-induced differentiation of human and rat bone marrow-derived MSCs into endothelial cells.

Probitics and antibiotics are all used as growth promoters in domestic animal breeding and poultry raising. In the present study, the effects of probiotics and antibiotics on structure and diversity of the intestinal microflora of broiler 817 was investigated. Ammonia and sulfured hydrogen in fecal were detected. Then the intestinal microflora was analyzed by the pour plate method and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. The results showed that the probiotics used significantly reduced the fecal emission of ammonia and sulfured hydrogen in broilers, and increased the number of lactic acid bacteria whereas antibiotics reduced it. Diversity analysis of cecal microflora by PCR-DGGE DNA fingerprinting revealed that the richness and variety of microflora in probiotics were obviously better than that in antibiotics group and control group. Key words: Probitics, intestinal microflora, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), diversity.

Myocardin-related transcription factor-A (MRTF-A) is a remarkably potent transcriptional co activator expressed in a broad range of embryonic and adult tissues. It shows that MRTF-A might induce cardiac hypertrophy in our previous studys. However, it remains poorly understood about the effects of MRTF-A in cardiac hypertrophy. Here, we show that the p300, a his tone acetyltransferase, enhanced transcriptional activity of MRTF-A. Conversely, class II his tone deacetylases HDAC5 suppress cardiac-special genes activation by MRTF-A and p300. These findings point that MRTF-A maybe a nexus for regulation of cardiac hypertrophic-special genes expression by changes in chromatin acetylation. Our results provide novel evidence to explore the roles of MRTFs and their co-factors in cardiac hypertrophy for the future studies.

Lactobacillus plantarum (L. plantarum) is a flexible and versatile microorganism that inhabits a variety of environmental niches, and some strains are marketed as probiotics. In this study, a strainTH1 which was isolated from silage in our former works was classified asL. plantarum by 16S rDNA sequence analysis and phylogenetic tree. Then its potential abilities of bile tolerance, nitrate reduction and anti-hypertension were investigated. The results showed that L. plantarum TH1 had capacity to resistance to bile salt, directly deoxygenate nitrate to nitrogen or ammonia rather then nitrite, and produce ACE inhibitory components in fermented milk. These works indicated that this novel lactobacillus strain might be considered as candidates for probiotics strains using in the food or drug system to improve the health of patients suffering from hypertension or other chronic disease.

The purpose of the experiment: to explore the primary cultural method of vascular endothelial cell in rat that we have got through obtained and cultivated, the experiment were provided to the corresp technique of the further research. Experimental methods: the aorta of Wistar rat was obtained in operating microscope, then removal the adventitia and adipose tissue. The blood vessel were sheared like the organization by 1mm×1mm. Using stationary culture in DMEM/F12 culture medium contained fetal bovine serum (FBS). The results of experiment: endothelial cell swimmed from the borderline of organization to grow by adherence after three days. Then the cell began to synthesis after 10 to 12 days and were confluenced to monolayer about 70%. The passage cell were grew prosperity from the digest of diastase vera and were converged like the cobblestone characteristic of endothelial cell. The method of experiment was convenient and economy, we can obtain vascular endothelial cell in high fineness without collagenase and the supplement of endothelial cell in cell growth.

Myocardin is a potent coactivator of SRF and expressed mainly in cardiomyocytes and smooth muscle cells (SMCs), Myocardin is important for SMC differentiation, but its precise role in regulating the initiation of SMC development is less clear. Constuction and function analysis of myocardin promoter luciferase reporter plasmid will provide the theory basis for researching the function of myocardin in SMC differentiation. In this study, a mouse myocardin promoter luciferase reporter construct was successfully constructed. To determine whether myocardin transcription activity were regulated by several factors, which play important roles in SMC differentiation, luciferase repoeter assays were performed in COS-7 cells and vascular smooth muscle cells (VSMCs). The results illustrated that Smad2 significantly activated myocardin promoter, but Smad3 signaling was inhibitory to myocardin expression at 18h. Myocardin promoter transactivity was inhibited by estrogen receptor α (ERα), and enhanced by p300. These findings indicated myocardin might be regulated by multi-signals and these factors might affect SMC differentiation through activating or inhibiting myocardin transcription.

Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor-β (TGF-β). However, the relationship or link between myocardin and TGF-β3-induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF-β3 were able to induce differentiation of rat bone marrow-derived MSCs toward smooth-muscle-like cell types, as evidenced by increasing expression of SMC-specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site-direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs. © 2012 IUBMB Life, 64(4): 331–339, 2012

The kringle 2 plus serine protease domains (K2S) of human tissue plasminogen activator (tPA) is an efficacious thrombolytic drug, which has been used to treat heart attacks and strokes by breaking up the clots that cause them. It has nine disulfide bridges, which are needed for proper folding and be the bottleneck in improving the production in the Escherichia coli system. So far, few reports have described the production of soluble active K2S from E. coli.To achieve high-level expression of active K2S in the E. coli system.The DNA fragment coding for K2S was fused with the E. coli disulfide isomerase DsbC. The constructed fusion protein was expressed in E. coli, and then purified with the Ni(2+)-chelating affinity chromatography. K2S was released by cleavage with Factor Xa protease, and the thrombolytic activity was determined using the fibrin plate assay.The fusion protein DsbC-K2S was found in the culture supernatant of recombinant E. coli as a soluble form of ~40%. The result of fibrinolysis fibrin plate assay showed that the purified recombinant K2S exhibited significant fibrinolysis activity in vitro.These works provided a novel approach for the production of active K2S in E. coli without the requirements of in vitro refolding process, and might establish a significant foundation for the following production of K2S.

A new compound, 3β,18,19β-trihydroxylupane, was isolated from Garcinia tetralata, along with five known compounds, garcinexanthone B, morolic acid acetate, toxyloxanthone A, 6,11-dihydroxy-2,2-dimethylpyrano[3,2-c]xanthen-7(2H)-one, and 1,4-dihydroxy-5,6-dimethoxy-xanthone. The structure of the new compound was established by extensive spectroscopic techniques.

Myocardin is a remarkably potent transcriptional coactivator expressed specifically in cardiac muscle lineages and smooth muscle cells during postnatal development. Myocardin shares homology with myocardin-related transcription factor-A (MRTF-A), which are expressed in a broad range of embryonic and adult tissues. Our previous results show that myocardin induces cardiac hypertrophy. However, the effects of MRTF-A in cardiac hypertrophy remain poorly understood. Our present work further demonstrates that myocardin plays an important role in inducing hypertrophy. At the same time, we find that overexpression of MRTF-A in neonatal rat cardiomyocytes might induce cardiomyocyte hypertrophy. Furthermore, MRTF-A expression is induced in phenylephrine, angiotensin-II, and transforming growth factor-β-stimulated cardiac hypertrophy, whereas a dominant-negative form of MRTF-A or MRTF-A siRNA strongly inhibited upregulation of hypertrophy genes in response to hypertrophic agonists in neonatal rat cardiomyocytes. Our studies indicate that besides myocardin, MRTF-A might play an important role in cardiac hypertrophy. Our findings provide novel evidence for the future studies to explore the roles of MRTFs in cardiac hypertrophy. © 2011 IUBMB IUBMB Life, 63(1):54–61, 2011.

Bacillus subtilis (B. subtilis) is a kind of Gram positive bacteria possessing multiple enzyme systems, which might be useful in wastewater treatment. In this paper, in order to confirm whether Bacillus subtilis BS7.29, a novel strain isolated from soil in our previous work, could be applied in the wastewater treatment, its effects on assimilation of ammonium, transformation of nitrite, and degradation of pectin were investigated. The results showed that the content of NH3-N and NO2-N in aquaculture water decreased with time increasing after being treated with B. subtilis BS7.29. Furthermore, when the secretion enzyme extracted from this bacterium was used to treat the paper-making white water, it could reduce CD and turbid it, decrease the size of colloidal substances and promote the deposition. These findings suggested that B. subtilis BS7.29 might be valuable in the wastewater treatment, and contribute to the development of biological purification of industrial effluents.

Recent studies have demonstrated that the expression of aquaporin1 (AQP1) in endothelia cells played important roles in cell migration, and migration-associated cell function such as wound healing, and neutrophil motility. In the present study, we provided evidence that AQP1 expression in vascular endothelia cells (VECs) during angiogenesis was associated with MRTF-A. Morphology and immunofluorescence assay demonstrated that VECs from normal and angiogenesis tissues had similar appearance. Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Immunoblot and Immunofluorescence analysis identified that AQP1 expression was significantly higher in angiogenesis VECs than in normal VECs. MRTF-A expression was also remarkably enhanced in angiogenesis VECs as compared to normal VECs, assessed by RT-PCR. The bioinformatics analysis found that the CarG element existed in the promoter region of AQP1 gene of many familiar mammals, including of mouse, rat, human etc. These results were the first to indicate that AQP1 was a target gene which could be regulated by MRTF-A/SRF.

Human Coagulation Factor IX (hFIX) plays a crucial role in the process of human endogenous blood clotting. Patients with deficiency of hFIX will suffer from hemophilia B. In this study, we use Pichia Pastoris to produce recombinant hFIX to cure hemophilia B Human liver hFIX cDNA were amplified by RT-PCR and inserted into pPIC9k vector, to construct pPIC9k-hFIX. The pPIC9k-hFIX were integrated into Pichia Pastoris SMD1168 chromosome by electro-transformation. Then we screened recombinant by PCR analysis and G418 selection to obtain high-copy recombinants. SDS-PAGE and Western Blot analysis show that protein expression volume is up to 100 mg/L. The protein was purifed and concentrated by anion exchange membrane diffusion dialysis. Then we assay activity by using lack coagulation factor IX. The activity calculation by standard curve show that the coagulation factor IX expressed by Pichia Pastoris has similar clotting activity (5.5%) compared to normal human plasma. Thus, expression of hFIX in Pichia Pastoris is of feasibility.