Inducer Research Articles

STRUCTURAL STUDIES OF TRANSCRIPTIONAL REGULATION BY LysR-TYPE TRANSCRIPTIONAL REGULATORS IN BACTERIA

LysR-type transcriptional regulators (LTTRs) comprise one of the largest families of transcriptional regulators in bacteria and control gene expression of various types of metabolic, virulence and physiological functions. LTTRs typically form homotetramers and require an inducer molecule(s) to activate the transcription of target genes. The N-terminal region of LTTRs contains a DNAbinding domain (DBD) with the winged helix-turn-helix motif that specifically binds the promoter region of target genes. The C-terminal region of LTTRs is connected to the DBD by a linker helix and forms the regulatory domain (RD) that contains a binding pocket for inducer molecules. Crystal structures of several LTTR family members together with their biochemical analyses have provided a potential mechanism for the initial process of transcriptional activation by LTTRs. First, helix α3 of the winged helix-turn-helix motif in DBD is supposed to distinguish the recognition binding site (RBS) in the promoter region, resulting in complex formation through interactions between two DBDs in the tetrameric LTTR and RBS. Formation of this complex seems to enable interactions between the other two DBDs in the LTTR tetramer and the activation binding site (ABS) in the promoter region. The binding of the tetrameric LTTR to both the RBS and ABS causes the promoter DNA to adopt a bent structure because the four DBDs in the tetrameric LTTR are arranged in a V-shaped manner at the bottom of the LTTR. Interaction of an inducer molecule(s) with the RD seems to cause a quaternary structural change of the LTTR that relaxes the bending angle of the promoter DNA with a concomitant shift of the bound DBDs at the ABS. These events facilitate recruitment of RNA polymerase to its binding site in the promoter region, which overlaps with the ABS for LTTR.

Evaluation of the simultaneous presence of aluminum chloride and 50 Hz magnetic field on the antioxidant capacity of blood hemoglobin and plasma in rat’s protective role of Myrtus plant

Introduction: Physical factors such as electromagnetic fields and chemical agents such as aluminum compounds can act as oxidative stress inducer. In the present study synergistic and toxic effects of aluminum in the presence of an alternating electromagnetic field is investigated. In order to make the protection the extract of Myrtus Communis was used Materials and Methods: In this experiment, there were 6 groups as follows: 1) control group 2) Aluminum chloride (8mg / kg) 3) the magnetic field (50 Hz and 1mT) 4) Aluminum chloride + magnetic field 5) Aluminum chloride + extract (1.5 mg/ kg) 6) A group that only receives the extract.7) magnetic field + aluminum chloride + extract. aluminum choloride was given intraperitoneally. The extract of Myrtus Communis (1.5 mg / kg) 2 hours before the injection by gavage was given to them. After experiments the blood samples were collected then Changes in the structure of hemoglobin, FRAP and MDA tests were performed Results: Exposure to electromagnetic field and aluminum chloride individually can reduce FRAP values (90.47±2.39, 73.36±1.89, 76.45±1.36, 68.27±1.32, 82.52±1.01, 80.22±1.44) (P <0.01) and increase in the amount of MDA(18.13±0.67, 12.25±0.19, 14.62±0.37, 8.54±0.57, 13.51±0.75, 12.38±0.35) (P <0.01). in case of aluminum chloride and magnetic field the synergistic effects of oxidative stress was enhanced (P <0.01). In all the above cases, the herbal extract can improve significantly the harmful effects (P <0.01) Conclusion: Each of Electromagnetic Fields and aluminum chloride can act as oxidizing agents, The combined effect of these two physical and chemical factors can act synergical and Cause biological damage.Treatment with extract Myrtus Communis can provide the protection against oxidative damage.

Effect of carbon monoxide postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration in rat hippocampal neurons: the relationship with mPTP/ROS signaling pathway

Objective To evaluate the effect of carbon monoxide(CO) postconditioning on pyroptosis induced by oxygen-glucose deprivation and restoration(OGD/R) in rat hippocampal neurons and the relationship with mitochondrial permeability transition pore(mPTP)/reactive oxygen species(ROS) signaling pathway. Methods Primary hippocampal neurons were cultured in vitro, seed in 6-well or 96-well plates, and divided into 5 groups(n=24 each) using a random number table method: control group(C group), OGD/R group, CO postconditioning group(CO group), specific mPTP opener atractyloside plus CO postconditioning group(ACO group), and specific ROS inducer antimycin A plus CO postconditioning group(KCO group). Neurons were subjected to O2-glucose deprivation(OGD) for 16 h followed by restoration of O2 -glucose supply for 24 h to establish the model of OGD/R injury. In group CO, neurons were exposed to 2% CO-5% CO2 for 3 h at 37 ℃ starting from the end of OGD, followed by normal culture for 21 h. In ACO and KCO groups, atractyloside 20 μmol/L and antimycin A 50 μmol/L were added at the end of OGD, respectively, and the other treatments were similar to those previously described in group CO. Neuronal pyroptosis rate was determined using double immunofluorescent staining cleaved caspase-1-AlexaFluor 568/DAPI after the end of treatments in each group. The neuronal survival rate was determined by MTT, opening of mPTP by Calcein-AM fluorescence, ROS content by DCFH-DA, and expression of interleukin-1beta(IL-1β) and IL-18 by Western blot. Results Compared with C group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in the other groups(P<0.05). Compared with OGD/R group, neuronal pyroptosis rate, ROS content and opening of mPTP were significantly decreased, the neuronal survival rate was increased, and the expression of IL-1β and IL-18 was down-regulated in CO, ACO and KCO groups(P<0.05). Compared with CO group, neuronal pyroptosis rate and ROS content were significantly increased, the neuronal survival rate was decreased, and the expression of IL-1β and IL-18 was up-regulated in ACO and KCO groups, and opening of mPTP was significantly inctreased in ACO group(P<0.05). Conclusion CO postconditioning can inhibit OGD/R-induced pyroptosis in rat hippocampal neurons, and the mechanism is related to inhibiting mPTP/ROS signaling pathway. Key words: Carbon monoxide; Neurons; Cell death; Reperfusion injury; Reactive oxygen species; Mitochondrial membrane transport proteins

Biochemical, physiological changes and antioxidant responses of cut gladiolus flower 'White Prosperity' induced by nitric oxide

Sodium nitroprusside (SNP), as nitric oxide (NO) donor, has been considered by postharvest researchers as one of the best option for slowing the processes controlling senescence in cut flowers. Here, we investigate the role of NO on postharvest physiology and vase life of the Gladiolus grandiflorus cv. White Prosperity. Vase life markedly extended by SNP at 150 μM from 3 day to 7.33 day and thus those inducer effects were dose- and time-dependent. SNP at 125 μM interdependent on vase life time period was observed to be the optimal dose for improving of relative fresh weight (RFW), peroxidase (POD), and total monomeric anthocyanin (TMA) in cut flowers. Supplementing vase solution with SNP indicated significant increase in water uptake of cut flowers and consequently protected to decline in RFW due to alleviate water losses stress. SNP was maintained the level of total soluble protein, lipid peroxidation, and POD, whereas it enhanced the level of catalase (CAT) and TMA in flower petals. Summary of our results revealed that SNP exogenous prolongs vase life via maintaining protein degrade, scavenging free radical in term of anthocyanin and enzymes antioxidant, decreasing polyphenol oxidase, inhibiting lipid peroxidation, and improving membrane stability in ‘White Prosperity’ cut flowers.

Effects of ADAR1 inducer / inhibitor on cognitive impairment and the expression of ADAR1 protein of social isolation mice

Objective To explore the effects of ADAR1 inducer and inhibitor on cognition and ADAR1 expression of isolated BALB/c mice. Methods Sixty healthy BALB/c mice were divided into 6 groups according to randomized design with 10 animals each group, the gregarious control group (GH), social isolation model group (SI), ADAR1 inducer treated gregarious group (GH+ IFN-γ), ADAR1inhibitor treated gregarious group (GH+ EHNA), ADAR1 inducer treated isolation group (SI+ IFN-γ) and ADAR1 inhibitor treated isolation group (SI+ EHNA). Mice in drug treatment groups were treated with ADAR1 inducer (5.0×104 U/kg, 20 ml/kg, ip) and inhibitor (10 mg/kg, 20 ml/kg, ip). Objection recognition test was used to measure cognition.Immunohistochenmistry was used to measure ADAR1 immunoreactivity and Western blot was used to measure ADAR1 protein expression. Results In the objection recognition test, the non-spatial discrimination index of mice in SI group (-0.16±0.09) was significantly lower than that of GH group (0.41±0.17, P<0.01), the non-spatial discrimination index of mice in SI+ IFN-γ group (0.20±0.09) and in SI+ EHNA group (-0.29±0.12) was higher (P<0.01) and lower (P<0.05) than that of the SI group respectively. The immunohistochemistry results showed that the ADAR1 immunoreactivity in hippocampus of mice in SI group (Hilus: (0.013±0.003), CA1: (0.021±0.005)) decreased significantly compared to those of GH group(Hilus: (0.021±0.002), (0.047±0.004); both P<0.05). And GH+ IFN-γgroup mice showed increased ADAR1 immunoreactivity obviously in Hilus (( 0.013±0.003) vs (0.023±0.004), P<0.01) and in CA1 ((0.021±0.005) vs (0.040±0.005), P<0.01) compared with that of SI group, ADAR1 inducer recovered the above abnornal ADAR1 immunoreactivity.Western blot results showed that the ADAR1 protein expression of mice in SI group (0.48±0.07) in hippocampus was significantly decreased (P<0.01) compared to that of GH group (1.00±0.00) .The level of ADAR1 protein in SI+ IFN-γgroup(0.82±0.04) increased compared with that of SI group. Conclusions Four weeks of social isolation can reduce the non-spatial cognitive ability of BALB/c mice and decrease the expression of ADAR1 in the hippocampus. The ADAR1 inducers and inhibitors can reverse and aggravate the cognitive impairment caused by social isolation respectively. The related mechanisms may be related to the expression of ADAR1. Key words: Social isolation; Cognitive function; BALB/c mice; Adenosine deaminase 1 acting on RNA

Expression of CD98 in colorectal cancer and its relationship with CD147

Objective To investigate the protein exp peopletracellular metalloproteinase inducer (CD147) in human colorectal cancer tissues. Methods EnVision immunohistochemical method was used to detect the expression of CD98 protein in 70 cases of colorectal cancer and their adjacent tissues. Western blotting was used to detect the expression of CD98 and CD147 protein in HCT116 cells after transfection with CD98 small interfering RNA (siRNA) and negative control. Results The positive rate of CD98 in colorectal cancer and the adjacent tissues was 57.1% (40/70) and 40.0% (28/70), which was statistically significant (P=0.042). The expression of CD98 in colorectal cancer was closely correlated with tumor stage (P=0.017) and lymph node metastasis (P=0.017), but not with gender (P=0.125), age (P=0.935), distant metastasis (P=0.464), tumor differentiation (P=0.995). There was a significant positive correlation between the expression of CD98 and CD147 in colorectal cancer (r=0.471, P=0.001). The expression of CD98 and CD147 protein was obviously decreased in HCT116 cells after transfection with CD98 siRNA, compared with negative control. Conclusion The expression of CD98 is closely related to the invasion and metastasis of colorectal cancer. CD98 may regulate the invasion and metastasis of colorectal cancer by up-regulating the expression of CD147. Key words: Colorectal cancer; CD147; CD98; Immunohistochemistry; Cell transfection

Shufeng Huoxue Formula suppresses proliferation and regulates melanin metabolism in murine B16 melanoma cells in vitro through autophagy pathway

To investigate the role of autophagy in the regulatory effect of Shufeng Huoxue Fumula (SFHXF) on the proliferation and melanin metabolism in cultured murine B16 melanoma cells. B16 cells were treated with solutions containing 0.12, 0.25, 0.49, 0.98, or 1.96 mg/mL SFHXF preparations, rapamycin (an autophagy inducer), or rapamycin+SFHXF. The changes in the proliferation of B16 cells were assessed using MTT assay, and tyrosinase activity and melanin content in the cells were determined. The expressions of autophagy-related proteins P62, p-mTOR, LC3B, and beclin 1 in the cells were detected using Western blotting. Compared with the blank control cells, treatments with SFHXF both in the presence and in the absence of rapamycin concentration-dependently inhibited the cell proliferation (P<0.05) and obviously increased tyrosinase activity and melanogenesis in B16 cells (P<0.05); 0.98 mg/mL SFHLF, rapamycin+0.98 mg/mL SFHXF, and 50 nmol/L rapamycin all significantly up-regulated the expressions of LC3B-II and beclin 1 and down-regulated the expressions of P62 and p-mTOR in the cells. SFHXF can regulate melanin metabolism and enhance tyrosinase activity and melanogenesis through the autophagy pathway to inhibit the proliferation of B16 cells in vitro.

Role of Nrf2 and NQO1 in the attenuation of oxygen-mediated pulmonary toxicity by sulforaphane

Background Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD). NrF2-mediated induction of NADPH Quinone oxydoreductase enzyme (NQO1) is known to protect the tissues from hyperoxic injury. Sulforaphane is a potent inducer of NrF2 and NQO1, and has been shown to improve cell survival under hyperoxic conditions. Objective To test the hypothesis that human pulmonary cells carrying specific SNPs on the human Nrf2 and NQO1 genes will display decreased inducibility by sulforaphane. Design/Methods: H441 cells …

The progress of TWEAK and its receptor Fn14 signal pathway in lung diseases

Tumor necrosis factor-like attenuated apoptosis inducer (TWEAK) is a new member of the tumor necrosis factor super family. TWEAK regulates cellular proliferation, differentiation, migration, apoptosis and inflammation by binding its receptor, fibroblast growth factor-inducible 14 (Fn14), through the interaction between cells or in a paracrine fashion. The role of TWEAK / Fn14 signaling pathway in the development of lung diseases such as lung injury, asthma and lung cancer has drawn increasing attention. Key words: TNF-related apoptosis-inducing ligand; Receptors, TNF-related apoptosis-inducing ligand; Review

The research of differentiation of skin-derived precursors into motor neurons induced by retinoic acid combined with Purmorphamine

Objective To explore the differentiation of skin-derived precursors (SKPs) into motor neurons (MNs) induced by retinoic acid (RA) combined with Purmorphamine (PM), and provide the evidence for motor neuron transplantation in the treatment of denervated muscle atrophy. Methods SKPs were isolated from the skin of neonatal rats, and cultured and proliferated in vitro. The expression of SKPs markers Nestin and Fibronectin was detected by immunocytochemistry. RA and PM were used as inducers to induce the differentiation of the third generation SKPs to MNs, and the growth factors such as GDNF and BDNF were added to promote the axon elongation and MNs maturity. The expression of MNs marker HB9 and choline acetyltransferase (ChAT) was detected by immunocytochemistry to identify whether the differentiated cells were MNs or not. The positive rate of ChAT and HB9 was calculated. Results The differentiated cells from SKPs induced by RA and PM had the morphology of MNs, and express the MNs specific markers HB9 and ChAT. The positive rate was (72.61±2.25)% for ChAT and (75.16±3.62)% for HB9. Conclusion The use of RA and PM can induce SKPs to differentiate into MNs. Key words: Motor neurons; Cell differentiation; Skin-derived precursors; Retinoic acid

Establishment of multidrug resistance cell line of cisplatin and gemcitabine in bladder cancer cell line T24 and its biological characteristics

Objective To establish a human bladder cancer cisplatin (DDP) and gemcitabine (GEM) resistant cell line (T24/DDP&GEM) by concentration gradient method, and to observe its biological characteristics. Methods DDP and GEM were used as inducers to establish human bladder cancer cisplatin and gemcitabine resistant model T24/DDP&GEM in vitro. The morphology of T24 and T24/DDP&GEM cells was observed by the inverted microscope. The drug resistance and the growth curve of cells were detected by cell counting kit-8 (CCK-8). The P-glycoprotein (P-gP) expression was detected by Western blotting. Results After the induction of DDP and GEM for 12 months, T24/DDP&GEM was successfully established. Under the inverted phase contrast microscopy, as compared with T24 cells, the morphology of T24DDP/GEM cells was irregular, the volume became larger, and vacuoles and granules were formed in the cytoplasm. The proliferation ability of T24/DDP&GEM cells was significantly lower than that of T24 cells. The doubling time of T24/DDP&GEM cells was (39.51±0.36) h, and that of T24 was (28.06±0.76) h. The doubling time was extended for (11.45±0.41) h. The drug resistance of T24/DDP&GEM was significantly improved, and the drug resistance index was 4.25 for DDP and 62.97 for GEM. As compared with T24 cells, the proportion of S and G2 phase cells in T24/DDP&GEM cells decreased, the proportion of G1 cells increased significantly, and the cell cycle slowed down. The results of Western blotting showed that the expression level of P-gp in T24/DDP&GEM cells was significantly higher than that in T24 cells. Conclusion The artificial induced DDP and GEM resistant bladder cancer cell line T24/DDP&GEM has the characteristics of multi drug resistant tumor cells. Key words: Bladder cancer; Chemotherapy; Cisplatin; Gemcitabine; Multidrug resistance

Expression of EMT inducers integrin-linked kinase (ILK) and ZEB1 in phyllodes breast tumors is associated with aggressive phenotype.

Phyllodes tumors (PTs) of the breast constitute an uncommon group of mammary fibroepithelial lesions with ambiguous biologic behavior. Recent evidence suggests that epithelial mesenchymal transition (EMT), a driving force of cancer progression is implicated in PTs pathogenesis. Integrin-linked kinase (ILK), a focal adhesion kinase, has been implicated in cancer and EMT and represents a novel cancer therapeutic target. In this study, we aimed to investigate ILK and EMT markers expression in phyllodes breast tumors in relation to tumor grade. Expression of ILK and EMT markers E-cadherin, β-catenin, Ν-cadherin, vimentin, Snail, ZEB1 and Twist was evaluated by immunohistochemistry in paraffin-embedded tissue sections from 96 human phyllodes breast tumors (48 benign, 27 borderline, 21 malignant). Cytoplasmic and nuclear immunopositivity of ILK were observed in both the epithelial and the stromal component of phyllodes breast tumors and were significantly higher with increasing tumor grade. An EMT-related expression profile consisting of decreased membranous and increased nuclear/cytoplasmic immunoreactivity of E-cadherin and β-catenin and increased expression of N-cadherin, vimentin, Snail, ZEB1 and Twist was observed in tumor epithelial and stromal component and was significantly associated with malignant phyllodes breast tumor histopathology. Interestingly, there was a significant correlation of ILK expression with all of the EMT markers examined. Our results suggest that EMT significantly contributes to phyllodes tumor pathogenesis and originally implicate ILK and ZEB1 in phyllodes tumors malignant phenotype.

The roles of cytochrome P450 and P-glycoprotein in the pharmacokinetics of florfenicol in chickens.

The effects of three selective oral inhibitors, fluvoxamine (FLU), ketoconazole (KET), and verapamil (VER), on the pharmacokinetics (PK) of florfenicol (FFC) were investigated in chickens. The chickens were administered orally with saline solution (SAL), FLU (60 mg/kg), KET (25 mg/kg), or VER (9 mg/kg) for 7 consecutive days. Florfenicol was given to the chickens at a single dose of 30 mg/kg orally. Blood samples were collected from each chicken at 0 to 12 h post-administration of FFC. The plasma concentration of FFC was analyzed by high-performance liquid chromatography (HPLC). The AUC of FFC increased and the CLs of FFC decreased with oral co-administration of KET in chickens, and the Cmax of FFC increased with VER. While the AUC, the CLs and the Cmax of FFC were all invariable with FLU. These data suggested that CYP 3A played a key role in the PK of FFC in chickens, however, P-glycoprotein (P-gp) and CYP 1A did not. The results imply that the adverse drug-drug interaction may occur in the use of FFC if the co-administrated drugs are the substrates, inducers or inhibitors of CYP 3A or/and P-gp.

Diagnosis and treatment of rheumatoid arthritis:toward the best practice. The Pathophysiology in Rheumatoid Arthritis.

Rheumatoid arthritis(RA)is one of the systemic autoimmune diseases characterized by progressive joint destruction with chronic synovitis. In the synovium, activated osteoclasts with plenty of inflammatory cytokines and metalloproteinases induce bone and cartilage destruction. Anti-cyclic citrullinated protein antibodies(ACPA)was recognized not only as a diagnostic marker but also as direct inducer of synovitis. In addition, environmental factors and background of daily life also contribute to the pathogenesis of RA, especially smoking and periodontitis are considered to be important factors for disease activities.

Fragility Fractures in Hemodialysis Patients. Abnormalities of osteocyte functions in chronic kidney disease.

Increased fracture incidence and ectopic calcifications which are accompanied with mineral metabolic disorders are well seen in dialysis patients. The role of bone in chronic kidney disease patients including dialysis has been investigated in clinical findings and experimental examinations. The abnormalities of osteocyte function have been pointed to as an active inducer of pathology of a deregulated endocrine organ.

Vascular Endothelial Growth Factor-A (VEGF-A) Single Nucleotide Polymorphisms and Endometriosis: Still a Controversial Issue:

Endometriosis is a chronic, multifactorial, polygenic gynecological disease. Endometrium has a high angiogenic potential and endometriotic lesions involve large areas with a rich blood supply. Angiogenesis is controlled by numerous inducers, including the vascular endothelial growth factor (VEGF) family. Endometrium undergoes cyclical growth and regression during the menstrual cycle, which depends on ovarian steroid levels and is a rich source of angiogenic growth factors, including VEGF-A. The VEGF-A gene is located on chromosome 6p21.3 and it is highly polymorphic with more than 20 different variants. This article critically reviews the published data concerning the relationships between some of the VEGF-A single nucleotide polymorphisms and the risk, pathogenesis and stage of endometriosis. Contrasting results have been published in the literature - probably due to the different ethnic background and the number of patients enrolled in clinical trials. However, the increasing interest in the use of anti...

Effects and mechanisms of up-regulation of heme oxygenase-1 on uric acid-induced adipocyte dysfunction

Objective To investigate the effects and mechanisms of up-regulation of heme oxygenase-1(HO-1)on uric acid-induced adipocyte dysfunction. Methods Human bone marrow-derived mesenchymal stem cells(MSCs)were cultured in vitro and second or third generation MSCs were selected and recultured in an adipogenic induction medium, with the addition of various amounts of fructose and uric acid to find the optimal concentrations.Then, the HO-1 inducer cobalt protoporphyrin(CoPP)and the HO-1 inhibitor tin porphyrin(SnMP)were added successively.There were five independent samples in each group.Adipogenesis in MSC-derived adipocytes and droplets were measured by spectrophotometry, expression levels of xanthine oxidase(XO)and NADPH were measured by Western blott, superoxide levels were measured by Luminous spectrometer, and levels of adipogenic markers and HO-1 were measured by reverse transcription-polymerase chain reaction(RT-PCR). Results Fructose at 500 μmol/L and uric acid at 50 mg/L were the optimal concentrations for stimulating adipogenesis in MSC-derived adipocytes(P<0.05). Compared with the controls, fructose significantly increased the levels of XO expression and uric acid(P<0.05), and concurrent treatment with fructose and CoPP effectively reversed both XO and uric acid levels to those of the controls(all P<0.05). However, SnMP negated the beneficial effects of CoPP(P<0.05). Additionally, uric acid decreased the number of small droplets and increased expression levels of adipogenic markers and NADPH(all P<0.05), but proadipogenic mediator levels were reduced with the addition of CoPP(all P<0.05). Furthermore, uric acid reduced expression levels of Wnt10b, but had no significant effect on HO-1 expression, andthese effects were reversed upon the addition of CoPP(all P<0.05). Conclusions Upregulation of HO-1 can reduce the levels of XO and uric acid, adipogenesis in MSC-derived adipocytes, and also the expression of adipogenic markers and NADPH.Therefore, it plays a role in antioxidant stress.HO-1 agonists may be targets for treating organ damage and controlling weight in elderly obese patients with metabolic syndrome. Key words: Heme oxygenase-1; Uric acid; Adipocytes; Oxidative stress

Simulated Microgravity Condition Alters the Gene Expression of some ECM and Adhesion Molecules in Adipose Derived Stem Cells.

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.

Induction of proteases secreted by fusarium oxysporum F. SP. Dianthi in the presence of carnation root cell walls. Biochemical characterization of a serine protease

We determined that protease activity is secreted by Fusarium oxysporum f. sp. dianthi race 2 (Fod) in vitro. The cell wall extracted from resistant and susceptible cultivars of carnation (Dianthus caryophyllus L.) was used as an inducer. The activity increased in the presence of the two cultivars, and higher levels were detected with the susceptible cultivar. One of the three proteases secreted by Fod was purified using ion exchange (DEAE-cellulose), gel filtration (Sephadex G-75), and affinity chromatographies (Benzamidine Sepharose 6B). Using protease inhibitors, the purified enzyme was classified as a trypsin-like serine protease that was inhibited by phenylmethanesulfonyl fluoride, diisopropylfluorophosphate and the soybean trypsin inhibitor. In addition, the enzyme was biochemically characterized and determined to have an optimal pH of 8.0, an optimal temperature of 50°C, stability from pH 6 to 8, temperature stability between 40 to 60°C, a molecular weight of 54 kDa, and an isoelectric point of 4.4. The values of the Km and Vmax kinetic constants were found to be 0.31 mg/ml and 24.7 µmol/min, respectively.

Role of CYP3A4 in kinase inhibitor metabolism and assessment of CYP3A4 activity

Tyrosine kinase inhibitors (TKIs) are small pharmacologically active molecules, which are administered orally at fixed doses to combat tumor cells. Although oral administration is advantageous, the variability of exposure is important for drug efficacy. Hence, the impact of individual CYP3A activity as one of the most important enzymes regulating exposure is important to know. To assess the relevance of CYP3A’s contribution to the clearance of TKIs, 31 TKI European public assessment reports (EPAR) were evaluated. AUC ratios (AUCR) from drug-drug interactions studies with ketoconazole (strong CYP3A inhibitor) and rifampicin (strong CYP3A inducer) were calculated and a significant linear regression was found between both AUCRs (r 2 =0.6951) after nonlinear regression analysis of the log transformed data. In general, if CYP3A contributes to more than 50% to the overall clearance, dose adaptations should be applied. Phenotyping CYP3A by midazolam microdosing might be an applicable option in future to adjust the TKI dose to the individual CYP3A activity, which should then be validated by therapeutic drug monitoring (TDM) to achieve optimal—efficacious but non-toxic—drug concentrations.