This research uses six Agrobacterium rhizogenes R1601, R15384, R1000, A4, R1025 and R1 to infect silymarin explants to induce hairy roots and silibin. All of the six A. rhizogenes can induce Silybum marianum to generate hairy roots and the A. rhizogene A4 shows comparatively high infection on the plant. This research determines the condition to induce silymarin hairy roots by the factors of infection time, pre-culturing, co-culturing and pH value. The fact that MS liquid medium fits the proliferation of silymarin hairy roots is determined. Through PCR molecular identification, it can be seen that the DNA plasmids in the A. rhizogenes are successfully integrated into the genome of transformed roots. Using liquid chromatography, it is determined that the silibin content in silymarin hairy roots is 2.5 times that in the plant In this research, the silymarin hairy roots culturing system is established, which lays a foundation for the study of culturing silymarin hairy roots and producing silibin.
Agrobacterium ; genetics ; physiology ; Cell Culture Techniques ; methods ; Milk Thistle ; chemistry ; genetics ; growth & development ; microbiology ; Plant Roots ; chemistry ; genetics ; growth & development ; microbiology ; Silymarin ; analysis ; Transformation, Genetic

Microtubule is one of the key components of the cytoskeleton and plays an important role in the maintenance of cell shape and the process of signal transduction and mitosis. Due to the extreme importance of microtubule in the process of mitosis, tubulin becomes one of the most important targets for development of new anticancer drugs and tubulin inhibitors are used for the treatment of cancer nowadays. These inhibitors have antitumor activity by inhibiting or promoting the assembly of tubulin to microtubules and interfering the process of cell mitosis. This review summarized the research progress of the tubulin inhibitors, especially the introduction of the tubulin inhibitors of pharmacological activities and the progress of clinical research. Also, the development trend of these inhibitors is discussed.

To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats.
Animals ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; pharmacokinetics ; Humans ; Isoenzymes ; genetics ; metabolism ; Liver Cirrhosis ; drug therapy ; enzymology ; genetics ; Male ; Mass Spectrometry ; Rats ; Rats, Wistar

In this study we present our experiences with the reverse sural fasciocutaneous flap to reconstruct the distal lower limb soft tissue defects caused by traumatic injuries. These flap graftings were carried out from Oct. 2010 to Dec. 2012 in our department. The series consisted of 36 patients, including 21 men and 15 women with an average age of 46.2 years (14-83 years) and with a medium follow-up period of 18 months (12-24 months). Of all the cases of acute trauma, there were 10 cases of trauma of distal tibia, 9 cases of trauma of perimalleolus, and 17 cases of trauma of midfoot and forefoot. Related risk factors in the patients were diabetes (2 cases), advanced age (>65 years, 3 cases) and cigarette smoking (6 cases). The reverse flow sural island flap irrigation depended on lower perforators of the peroneal artery. The fasciocutaneous pedicle was 3-4 cm in width and the anatomical structures consisted of the superficial and deep fascia, the sural nerve, short saphenous vein, superficial sural artery together with an islet of subcutaneous cellular tissue and skin. The most proximal border of the flap was only 1.5 cm away from the popliteal skin crease and the pivot point was 5-7 cm above the tip of the lateral malleolus. All the flaps survived. No arterial crisis occurred in any case. The venous congestion occurred in 2 cases and got better after raising the limbs and bloodletting. Only in an old man, 1.5 cm necrosis of distal margin of his flap occurred and finally healed after continuous dressing change. One-stage skin grafting was performed, and all the donor sites were sutured and successfully healed. It was concluded that the reverse sural fasciocutaneous flap is safe and reliable to extend to the proximal third even near the popliteal skin crease. We also concluded this flap can be safely and efficiently used to treat patients with large and far soft-tissue defects from the distal leg to the forefoot with more versatility and it is easier to reach the recipient sites.

Abstract: Toxoplasma gondii, an opportunistic pathogenic protozoan, is widely distributed worldwide and can cause zoonoses, which is a serious threat to human health. Nowadays, the relationship between T. gondii infection and neuropsychiatric diseases has attracted researchers' attention increasingly. T. gondii infection is related to the pathogenesis of many neuropsychiatric diseases by affecting the nervous system, such as schizophrenia, depression, Alzheimer's disease, and so on. This review will focus on the relationship between T. gondii infection and neuropsychiatric diseases and summarizes the possible mechanisms of disorders resulting from T. gondii infection． It is expected that the study on the related pathogenic mechanism of T. gondii will lead to new therapeutic directions and feasible solution for the clinical treatment of neuropsychiatric diseases caused by T. gondii infection.

OBJECTIVETo establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte).

METHODSBased on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated.

RESULTSWe have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.

CONCLUSIONSThe nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.

DNA, Circular ; genetics ; DNA, Viral ; genetics ; Hepatitis B ; diagnosis ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Leukocytes, Mononuclear ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

BACKGROUNDHepatocellular carcinoma (HCC) ranked the second among the causes of cancer mortality in China since the 1990s. Up to now, medication still plays an important role in the treatment of HCC. The therapies based on the allicin as a potential chemopreventive analog although is in its infancy at the present time, may have a significant role in the future management of HCC. Diallyl trisulfide (DATS) is a natural compound derived from garlic. In this study, we investigated the inhibitory effects of hepatic targeted polybutylcyanoacrylate nanoparticles of diallyl trisulfide (DATS-PBCA-NP) on orthotopic transplanted HepG2 hepatocellular carcinoma in nude mice.

METHODSDATS-PBCA-NP were detected by transmission electron microscope (TEM) and high-performance liquid chromatography (HPLC). The orthotopic transplantation HCC models were established by implanting HCC HepG2 xenograft bits under the envelope of the mice liver. Successful models (n = 29) were divided into 4 groups: normal saline (NS), empty nanoparticles (EN), DATS and DATS-PBCA-NP were intravenously administered to the mice respectively for 2 weeks. In vivo antitumor efficacy was evaluated by the measurement of tumor volume. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and protein levels of apoptosis and cell proliferation proteins by immunoblotting in tumor tissues were performed to elucidate the possible mechanism.

RESULTSDATS-PBCA-NP possessed smooth and round appearance, dispersed well, and released in vitro in accord with double phase kinetics model. DATS-PBCA-NP changed the tissue/organ distribution of DATS in vivo. The successful rate of tumor implantation was 100%. Intravenous administration of DATS-PBCA-NP significantly retarded the growth of orthotopically transplanted hepatoma in BALB/c nude mice (compared with the other three groups, all P < 0.05) without causing weight loss (P > 0.05). TUNEL staining showed that the tumors from DATS-PBCA-NP treated mice exhibited a markedly higher apoptotic index compared with control tumors. Western blot analysis of tumor tissue revealed that the down-regulated expression of proliferation cell nuclear antigen (PCNA) and Bcl-2 proteins in DATS-PBCA-NP group, and there were no significant differences in the expression of Fas, FasL and Bax proteins among the four groups (P > 0.05).

CONCLUSIONSDATS-PBCA-NP has good prolonged release effect in vivo and hepatic-targeted activity, and significant anti-tumor effect on the orthotopic transplantation HCC model in mice in association with the suppression of proliferation and the induction of apoptosis of tumor cells. These advantages are probably due to their liver targeting characteristics and consequently bring a higher anti-tumor activity.

Allyl Compounds ; administration & dosage ; Animals ; Antineoplastic Agents ; administration & dosage ; Apoptosis ; drug effects ; Blotting, Western ; Cell Line, Tumor ; Down-Regulation ; Enbucrilate ; administration & dosage ; Humans ; Liver Neoplasms, Experimental ; drug therapy ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nanoparticles ; Neoplasm Transplantation ; Proliferating Cell Nuclear Antigen ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Sulfides ; administration & dosage ; Transplantation, Heterologous

The present study was aimed to investigate the effects of hypoxia on iron metabolism of skeletal muscle. Rat L6 skeletal muscle cells were randomly divided into three groups which were exposed to hypoxia (1% O(2)) for 0, 12, 24 h, respectively. Iron isotope tracing method was used to determine iron uptake and release. Iron content of labile iron pool (LIP) was investigated by flow cytometry, and the expressions of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hypoxia-inducible transcription factor-1 (HIF-1) in L6 cells were observed by Western blot. The results showed that, compared with 0-hour hypoxia group, 12-hour hypoxia group exhibited significantly increased iron uptake and LIP (P < 0.05), as well as decreased iron release (P < 0.01). Not only iron uptake and release, but also LIP in 24-hour hypoxia group were significantly decreased, compared with those in 0- and 12-hour hypoxia groups (P < 0.01). The expressions of HIF-1, DMT1 (IRE), DMT1 (non-IRE) and TfR1 in 12-hour hypoxia group were significantly increased compared with those in 0-hour hypoxia group (P < 0.01). On the contrary, the expressions of DMT1 (IRE), DMT1 (non-IRE) and FPN1 in 24-hour hypoxia group were significantly decreased compared with those in the other two groups. However, TfR1 expression in 24-hour hypoxia was higher than those in 0- and 12-hour hypoxia groups (P < 0.05 or P < 0.01). These results suggest hypoxia plays an important role in iron metabolism of skeletal muscle cells. Moderate hypoxia can increase iron uptake and decrease iron release, resulting in higher LIP, but a prolonged hypoxia induces a disordered iron metabolism of skeletal cells.
Animals ; Cation Transport Proteins ; metabolism ; Cell Hypoxia ; Cell Line ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Iron ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Oxygen ; physiology ; Rats ; Receptors, Transferrin ; metabolism ; Time Factors

OBJECTIVETo investigate the role and significance of ATP-sensitive K+ channels in the pathological process of hypoxia hypercapnia-induced pulmonary vasoconstriction (HHPV) and the relationship with ERK1/2 signal pathway in rats.

METHODSWe made the third pulmonary artery rings of SD rats, used the model of pulmonary artery rings perfusion in vitro. Under acute hypoxia hypercapnia condition, and observed the effects of the three stages of HHPV incubated by glybenclamide(Gly) and the combined application of Gly and U0126. At the same time, the values of rings' tension changes were recorded via the method of hypoxia hypercapnia conditions reactivity.

RESULTSUnder the normoxia condition, the values of the third pulmonary artery rings tension were relatively stable, but under the hypoxia hypercapnia condition, we observed a biphasic pulmonary artery contractile response compared with N group (P < 0.05, P < 0.01). When the third pulmonary artery rings incubated by Gly, it's phase II persistent vasoconstriction was enhanced compared with the H group (P < 0.05, P < 0.01), and the phase I vasoconstriction was also heightened. Moreover, under the hypoxia hypercapnia condition, U0126 could significantly relieve the phase II persistent vasoconstriction compared with HD group (P < 0.05, P < 0.01) induced by Gly, but the phase I acute vasoconstriction and the phase I vasodilation had no changes (P > 0.05).

CONCLUSIONGly may mediate HHPV via activating ERK1/2 signal transduction pathway.

Animals ; Glyburide ; pharmacology ; Hypercapnia ; metabolism ; physiopathology ; Hypoxia ; metabolism ; physiopathology ; In Vitro Techniques ; MAP Kinase Signaling System ; physiology ; Male ; Pulmonary Artery ; drug effects ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; drug effects

The aim of this study was to understand the enterovirus types and biological features of pediatric cases of HFMD in Sanmenxia City during 2011, and compare the latter to a cohort of healthy children. Stool samples of 55 cases of HFMD and 60 healthy children were collected for the isolation and identification of enteroviruses using RNA extraction and real-time RT-PCR assays. EV71 and CA16 were identified by nucleotide sequencing using virus-specific VP1 primers; for the other enteroviruses, 012/011 and 008/013 primers were used for amplification and sequencing. The results were analysed by sequence alignment with known sequences, and the characteristics of the EV71 VP1 gene were also analyzed. The detection rates for enteroviruses in cases of HFMD and healthy children were 52.73% (29/55) and 18.33% (11/60), respectively. Among these, there were 22 cases of EV71, four cases of CA16 and three cases of other enteroviruses in the cases with HFMD. Eleven healthy children had intestinal viruses, of which nine were Coxsackie B virus strains (81.82%, 9/11). Gene sequencing of the 19 EV71 strains illustrated that they were all subgenotype C4a, but the evolutionary tree showed an obvious clustering between cases from Lingbao City and Lushi County. This study demonstrates that the EV71 subgenotype C4a and CA16 strains were the most common cause of HFMD in Sanmenxia City in 2011, and that Coxsackie B strains were prevalent in healthy children. This finding may indicate that there is a widespread source of recessive infection in the community.
Child ; Child, Preschool ; China ; epidemiology ; Cities ; epidemiology ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; Female ; Hand, Foot and Mouth Disease ; epidemiology ; virology ; Humans ; Infant ; Male ; Phylogeny ; Viral Proteins ; genetics