Fosmidomycin (100 micromol x L(-1)) which is the effective inhibitor of DXR, key enzyme in terpenoid MEP pathway, was used to treat with hairy roots of Salvia miltiorrhiza. The treated roots were harvested at 2, 4, 6, 8, 10, 16 and 21 d, mRNA level of SmDXR and tanshinone content in treated and negative control groups were detected. Results found that, after treated with fosmidomycin, color of S. miltiorrhiza hairy roots grew pale gradually comparing with controls; mRNA level of SmDXR in hairy roots varied as a shape of parabolic and the highest value achieved at the sixth day after treatment, then it decreased gradually; Content of four kinds of tanshinones were detected. Among of the four kinds of tanshinones, Tanshinone I content changed relatively little, while content of dihydrotanshinone I, cryptotanshinone and tanshinone II (A) decreased gradually in 21 days. The content of total tanshinones in NC groups was 5, 63 times more than FOS-treated roots in the 21th day. The previous results showed that SmDXR played an important role in the accumulation of tanshinone content in MEP pathway. Once the mRNA level of SmDXR was suppressed, the accumulation of secondary metabolites will be significantly affected.
Aldose-Ketose Isomerases ; genetics ; Diterpenes, Abietane ; metabolism ; Fosfomycin ; analogs & derivatives ; pharmacology ; Gene Expression Regulation, Plant ; drug effects ; Plant Roots ; drug effects ; growth & development ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; growth & development ; metabolism ; Time Factors

Worldwide sales of biologic drugs exceeded 100 billion USD in 2011. About 32% is from therapeutic monoclonal antibody (mAb). With many blockbuster biopharmaceutical patents expiring over the next decade, there is a great opportunity for biosimilar to enter the worldwide especially emerging market. Both European Medicines Agency (EMA) and Food and Drug Administration (FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics. Rather than providing a highly abbreviated path, as in the case for small molecule chemical drug, approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis. Since mAb is the dominant category of biologic drugs, mAb will be the focus of this review. First, the United States (US) and European Union (EU) approved mAb and those in phase 3 trials will be reviewed, then strategies on how to win biosimilar competition will be reviewed.
Animals ; Antibodies, Bispecific ; therapeutic use ; Antibodies, Monoclonal ; therapeutic use ; Antibodies, Monoclonal, Humanized ; therapeutic use ; Biosimilar Pharmaceuticals ; standards ; Clinical Trials, Phase III as Topic ; Drug Approval ; European Union ; Humans ; United States ; United States Food and Drug Administration

OBJECTIVETo study the roles of IL-4, IL-5 and IgE in childhood cough variant asthma (CVA).

METHODSThe IL-4 and IL-5 levels in peripheral blood mononuclear cell (PBMC) and the serum IgE levels were determined using ELISA in children with CVA in the acute stage (n=21) and in the convalesce stage (n=9). The samples from 30 children with acute bronchial asthma and from 30 healthy children were used as controls.

RESULTSThe levels of PBMC IL-4 (91.57 +/- 12.19 ng/L) and IL-5 (13.28 +/- 0.31 ng/mL) in children with CVA in the acute stage were significantly higher than those in the convalesce stage (74.68 +/- 11.54 ng/L, 6.53 +/- 0.28 ng/mL) and also higher than those in the healthy controls (70.32 +/- 18.16 ng/L, 5.29 +/- 0.36 ng/mL) (P < 0.01). The levels of serum IgE in children with CVA in the acute stage (279.6 +/- 41.3 KU /L) were strikingly higher than those in the convalesce stage (153.8 +/- 37.5 KU/L) (P < 0.01). The levels of serum IgE in children with CVA either in the acute stage or in the convalesce stage were significantly higher than those in healthy controls (90.6 +/- 44.8 KU /L) (P < 0.01). There were no significant differences in the levels of IL-4, IL-5 and IgE between children with acute CVA and acute asthma.

CONCLUSIONSA combined determination of PBMC IL-4 and IL-5 and serum IgE may be valuable for the diagnosis and the outcome evaluation of CVA. IL-4 and IL-5 may play a role in the pathogenesis of CVA. It is speculated that CVA may have similar pathogenesis to bronchial asthma since acute CVA patients have similar IL-4, IL-5 and IgE levels to children with acute bronchial asthma.

Asthma ; immunology ; Child, Preschool ; Cough ; immunology ; Female ; Humans ; Immunoglobulin E ; blood ; Infant ; Interleukin-4 ; blood ; physiology ; Interleukin-5 ; blood ; physiology ; Male

Objective To explore the relationship between intracellular calcium levels ([Ca2+]1) and reactive oxygen species (ROS) in sodium fluoride (NaF)-induced injury in human neuroblastoma SH-SY5Y cells. Methods The levels of [Ca2+]1 and ROS were measured in different exposed times(0,3,6,12,18,24 h) respectively after SH-SY5Y cells were exposed to 40 mg/L NaF in vitro, and the optimal expose time was selected. Furthermore, the changes of [Ca2+]1, ROS and LDH levels in 40 mg/L NaF-treated groups incubated with 38.23 mg/L BAPTA-AM or 380.40 mg/L ethylene glycol-bis-(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA) or 16.32 mg/L N-acetyl-L-cysteine(NAC) also observed at the optimal expose time(12 h), respectively. Results At 3,6,12,18 and 24 h, [Ca2+]1 level(5620.0±226.3,4775.5±85.6,3312.3±87.5, 3047.0±75.0,2717.0±66.5) was significantly increased, and so was the ROS level(4449.53±324.61,7463.07±117.43,20 227.33±178.04,8817.56±200.13, 7975.61±92.90) except at 3 h, compared with 0 h(2115.0±24.0,4098.01±21.22, all P<0.05). The levels of [Ca2+]1 and ROS reached the peak at 3 h and 12 h, respectively. [Ca2+]1 and LDH levels in NaF-treated group [3279.5±94.0, (1057.50±64.35)U/L], NaF+NAC treated group[ 3583.0±350.7, (561.02±85.50)U/L], NaF+EGTA treated groups[3701.5±157.7, (1074.50±86.97)U/L], and BAPTA-AM treated group[2766.5±38.9, (521.43±40.80)U/L] had increased, compared with the control[2022.5±118.1, (186.97±8.73)U/L], the difference being statistically significant (P<0.05). ROS levels in NaF-treated group (19 003.04±332.34), and NaF+EGTA treated group(19 170.12±95.46) was higher than that in the controls(4060.98±145.66), the difference being statistically significant (P<0.05). NaF and NAC had antagonistic effect on ROS and LDH levels (F=976.11,43.54,P<0.05). And NaF and BAPTA-AM had antagonistic effect on [Ca2+]1, ROS and LDH levels (F=15.65,1515.53,115.00, P<0.05). Conclusions NaF-related calcium is released from the site of intracellular calcium storage, which induces ROS production, both of them caused cytotoxicity and the increase of LDH level in human neuroblastoma SH-SY5Y cells.

AIMTo determine whether the cardioprotection of ischemic postconditioning and heptanol in ischemic heart against ischemia/reperfusion (I/R) is mediated by gap junction.

METHODSThe effect of ischemic postconditioning, heptanol at different doses (0.03, 0.06, 0.30, and 0.60 mg/kg) and AAP10 (10 mg/kg) on the intact rat heart during 30 min ischemia and 2 h of reperfusion was observed. Ischemic postconditioning was achieved by 3 cycles of 10 s reperfusion/10 s regional ischemia starting at the beginning of the reperfusion. The infarct size and the arrhythmia scores were measured. The effect of ischemic postconditioning, heptanol at different doses (0.05, 0.10, 0.50 and 1.00 mmol/L) and AAP10 (1 x 10(-7)mol/L) on the isolated heart during 30 min ischemia and 2 h of reperfusion was observed. Ischemic postconditioning was achieved by 6 cycles of 10 s reperfusion/10 s global ischemia starting at the beginning of the reperfusion. The arrhythmia scores and conduction velocity of ventricle muscle were measured.

RESULTSIn the intact rat heart model, ischemic postconditioning and heptanol reduced infarct size and arrhythmia scores. In the Langendorff perfused rat heart model, ischemic postconditioning and heptanol reduced arrhythmia scores and conduction velocity of ventricle muscle. Administration of AAP10, an opener of gap junction attenuated the cardioprotection of ischemic postconditioning and heptanol.

CONCLUSIONThe cardioprotection of ischemic postconditioning and heptanol may be related to the attenuation of gap junction communication on myocardiac ischemia/reperfusion injury.

Animals ; Gap Junctions ; physiology ; Heptanol ; pharmacology ; Ischemic Postconditioning ; methods ; Male ; Myocardial Ischemia ; physiopathology ; Myocardial Reperfusion Injury ; physiopathology ; prevention & control ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the best methods and skill for the removal of difficult and high risk tracheobronchial foreign body under bronchoscope.

METHODSA retrospective review was performed between August 1995 to August 2012. There were 4217 children with tracheobronchial foreign body, among them, 272 were diagnosed as high-risk, highly difficult tracheobronchial foreign bodies confirmed by clinical manifestations, foreign body type and bronchoscopy.

RESULTSIn 271 children, the tracheobronchial foreign body was removed under bronchoscope, the success rate was 99.6%; only one child with a pen cap blocking the left lower lobe bronchus was transferred to the department of thoracic surgery, and the foreign body was finally removed by thoracotomy. Eighty-five children (among them, 82 children were under 1 year of age) had II-II degree laryngeal obstruction, the emergency surgery was performed to remove the foreign body and to relieve the laryngeal obstruction. Twenty-six children had lung infection and 27 children had failed foreign body removal surgery before, in all these children, the foreign body was removed after infection control. There were 17 children with the pen cap as the tracheobronchial foreign body, direct removal was successful in 12 children with the history less than two weeks; in 4 children, the foreign body was removed after 0.1% epinephrine saline flush, and 1 case with the homemade bronchial foreign body hook remove. There were 26 children with the whistle as the foreign body, and 32 children had large and sharp foreign bodies. In these cases, the foreign bodies were removed together with the bronchoscope. Forty-two children had multiple or fragile foreign bodies, and 16 children had subsegmental bronchial foreign bodies. In these cases, the foreign bodies were removed with forceps under direct vision and intraoperative bronchial lavage.In This series, 129 children received intraoperative bronchial lavage, among them, 127 children showed normal X-ray changes one week after operation. Two children with a history of more than 1 month complicated with pulmonary consolidation. After bronchial lavage, pneumothorax and subcutaneous emphysema occurred, which recovered after treatment. No glottic edema, asphyxia, and other complications were found, the complication rate of surgery was 0.7%.

CONCLUSIONFor the removal of highly difficult and high risk tracheobronchial foreign bodies, preoperative analysis and discussion should be sufficient, appropriate surgical skill and surgical instruments may improve the success rate of the surgery and prevent the operation complications.

Adolescent ; Bronchi ; Bronchoscopy ; methods ; Child ; Child, Preschool ; Female ; Foreign Bodies ; surgery ; Humans ; Infant ; Male ; Retrospective Studies ; Trachea

OBJECTIVETo explore whether the inhibitory effect of Genipin on uncoupling protein-2 (UCP-2) in mitochondria is involved in energy metabolism of androgen-independent PC3 prostate cancer cells.

METHODSPC3 prostate cancer cells were cultured and treated with Genipin at the concentrations of 40, 80, and 160 μmol/L for 48 hours. Then the proliferation of the cells was detected by MTT assay, the expression of UCP-2 mRNA determined by RT-PCR, and the content of intracellular pyruvic acid (PA) and the activity of succinate dehydrogenase (SDH) in the mitochondria measured by visible spectrophotometry.

RESULTSWith the increased concentration of Genipin, the proliferative activity of the PC-3 cells, the expression level of UCP-2 mRNA, the content of intracellular PA and the activity of SDH in the cells were all decreased, namely, with the enhanced inhibitory effect of Genipin on UCP-2, a trend of reduction was observed in the proliferation of the cells, intracellular PA content, and SDH activity in the mitochondria.

CONCLUSIONGenipin is involved in the energy metabolism of androgen-independent PC3 prostate cancer cells by reducing the content of intracellular PA and the activity of SDH in the mitochondria, which may be associated with its inhibitory effect on UCP-2.

Cell Line, Tumor ; drug effects ; Energy Metabolism ; Humans ; Ion Channels ; metabolism ; Iridoids ; pharmacology ; Male ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; Pyruvic Acid ; metabolism ; RNA, Messenger ; Succinate Dehydrogenase ; metabolism ; Uncoupling Protein 2

An expression system is described for high-yield production of recombinant soluble human FasL (shFasL) in Dictyostelium discoideum cells. DNA encoding amino acids 141 - 281 of hFasL was PCR amplified from cDNA derived from activated human neutrophils. The resulting product was fused with a DNA fragment encoding hCG-beta signal peptide and cloned in the expression vector pMB12neo. Dictyostelium strain AX3 was transfected with this plasmid, yielding a recombinant strain called AX3-pCESFL95-H3. In order to improve the shFasL expression level, pMB12neo was optimized by replacing its transcriptional terminator/ polyadenylation segment of the 2H3 gene with an actin8 terminator/polyadenylation segment, yielding derived expression vector pMB74. The recombinant Dictyostelium strain called AX3-pLu8 was generated with this new plasmid. When the recombinant cells were cultivated in a complex HL-5C medium, a cell density of (1.5 - 2) x 10(7)/mL was reached, and the shFasL level expressed by strains AX3-pCESFL95-H3 and AX3-pLu8 was 23.5 microg/L and 206 microg/L, respectively. By using a newly developed synthetic medium called SIH as culture medium, higher cell density of (4 - 5) x 10(7)/mL was achieved. Correspondently, 111 microg/L and 420 microg/L shFasL were secreted by recombinant strains AX3-pCESFL95-H3 and AX3-pLu8, respectively.
Animals ; Chorionic Gonadotropin, beta Subunit, Human ; genetics ; Culture Media ; Dictyostelium ; genetics ; growth & development ; metabolism ; Fas Ligand Protein ; biosynthesis ; genetics ; Humans ; Neutrophils ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

To study the chemical constituents from the root of Berchemia lineata (L.) DC., nine compounds were isolated from the EtOAc extract by using silica gel, RP-C18 silica gel column chromatography and preparative HPLC. Based on the spectroscopic analysis, their structures were identified as 5-hydroxy-7-(2'-hydroxypropyl)-2-methyl-chromone (1), (-)-(1'R, 2'S)-erythro-5-hydroxy-7-(1', 2'-dihydroxypropyl)-2-methyl-chromone (2), naringenin (3), eriodictyol (4), (+)-aromadendrin (5), (+)-taxifolin (6), (+)-catechin (7), (+)-epigallocatechin (8) and quercetin (9). Among them, compound 2 is a new chromone derivative. Compound 1 is a known chromone derivative and isolated from this genus for the first time. Compounds 3-9 are known flavonoids and isolated from this plant for the first time.
Catechin ; analogs & derivatives ; chemistry ; isolation & purification ; Chromones ; chemistry ; isolation & purification ; Flavanones ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Molecular Structure ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quercetin ; analogs & derivatives ; chemistry ; isolation & purification ; Rhamnaceae ; chemistry

OBJECTIVETo study the accumulation of fluoride in rat hippocampus and its effect on cholinesterase activity.

METHODSRats were subchronically exposed to NaF, and fluoride concentration and cholinesterase activity in rat hippocampus were determined.

RESULTSFluoride concentration in rat hippocampus was significantly correlated with the dosage of fluoride, and there were significant differences among high dosage group [(13.03 +/- 1.79) micro g/g], low dosage group [(9.83 +/- 0.92) micro g/g] and control [(8.27 +/- 1.11) micro g/g], P < 0.01. Acetylcholinesterase activities among three groups [(0.111 +/- 0.031) micro mol/mg, (0.143 +/- 0.025) micro mol/mg, (0.183 +/- 0.027) micro mol/mg] were also significantly different (P < 0.01), which was negatively correlated with fluoride concentration in rat hippocampus (r = -0.700, P < 0.01). The activity of butylcholinesterase in high dosage group [(0.041 +/- 0.010) micro mol/mg] was different from that of control [(0.067 +/- 0.025) micro mol/mg, P < 0.05], but the activity was not significantly related with fluoride concentration in rat hippocampus (r = -0.317, P = 0.094).

CONCLUSIONFluoride may go through the blood-brain barrier and accumulate in rat hippocampus, and inhibit the activity of cholinesterase.

Acetylcholinesterase ; metabolism ; Animals ; Blood-Brain Barrier ; Butyrylcholinesterase ; metabolism ; Fluoride Poisoning ; metabolism ; Fluorides ; pharmacokinetics ; Hippocampus ; metabolism ; Male ; Organ Size ; Rats ; Rats, Sprague-Dawley