Quercetin inhibits truncated isoform of dopamine-and cAMP-regulated phosphoprotein as adjuvant treatment for trastuzumab therapy resistance in HER2-positive breast cancer

Trastuzumab resistance is one of the causes of poor prognosis in patients with human epidermal growth factor receptor 2(HER2)-positive(HER2+)breast cancer(BC).The truncated isoform of dopamine-and cAMPregulated phosphoprotein(t-DARPP)has been reported to be involved in trastuzumab therapy resistance and promoting tumor progression.To evaluate the t-DARPP expression in BC,paired tumors and surrounding normal tissues were analyzed by real-time polymerase chain reaction and confirmed higher DARPP-32 kDa family mRNA expression in HER2+BC tumor tissues.We established 2 patient-derived xenografts(PDX)mice models to test the efficacy of trastuzumab,named model 1(non-responder)and model 2(responder).t-DARPP and p95-HER2 protein-protein interactions were detected in PDX tumor tissue from non-responders using Förster resonance energy transfer assays.Instead,there is no response from the responder.Furthermore,mechanistic studies using transwell and western blot assays demonstrated that t-DARPP could upregulate epithelial-mesenchymal transition signaling proteins,enhance p95-HER2 expression and promote cell migration.We found that quercetin effectively reduced t-DARPP expression in HER2+BC cells.In t-DARPP ShRNA-suppressed cells,quercetin synergistically enhanced trastuzumab-induced apoptotic cell death and G2/M phase arrest.In conclusion,the combination of quercetin and trastuzumab treatment by targeting t-DARPP in HER2+BC patients has the potential as a biomarker for mitigating drug resistance.

Aldo-keto reductase family member C3(AKR1C3)promotes hepatocellular carcinoma cell growth by producing prostaglandin F2α

Hepatocellular carcinoma(HCC)is a leading cause of death worldwide.Current therapies are effective for HCC patients with early disease,but many patients suffer recurrence after surgery and have a poor response to chemotherapy.Therefore,new therapeutic targets are needed.We analyzed gene expression profiles between HCC tissues and normal adjacent tissues from public databases and found that the expression of genes involved in lipid metabolism was significantly different.The analysis showed that AKR1C3 was upregulated in tumors,and high AKR1C3 expression was associated with a poorer prognosis in HCC patients.In vitro,assays demonstrated that the knockdown of AKR1C3 or the addition of the AKR1C3 inhibitor indomethacin suppressed the growth and colony formation of HCC cell lines.Knockdown of AKR1C3 in Huh7 cells reduced tumor growth in vivo.To explore the mechanism,we performed pathway enrichment analysis,and the results linked the expression of AKR1C3 with prostaglandin F2 alpha(PGF2a)downstream target genes.Suppression of AKR1C3 activity reduced the production of PGF2a,and supplementation with PGF2a restored the growth of indomethacin-treated Huh7 cells.Knockdown of the PGF receptor(PTGFR)and treatment with a PTGFR inhibitor significantly reduced HCC growth.We showed that indomethacin potentiated the sensitivity of Huh7 cells to sorafenib.In summary,our results indicate that AKR1C3 upregulation may promote HCC growth by promoting the production of PGF2α,and suppression of PTGFR limited HCC growth.Therefore,targeting the AKR1C3-PGF2a-PTGFR axis may be a new strategy for the treatment of HCC.

Ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis

Objective： To examine the apoptotic effect of ent-llα-hydroxy-15-oxo-kaur-16-en-19-oic-acid （5F）, a compound isolated from Pteris semipinnata L （PsL）, in human lung cancer A549 cells. Methods： A549 cells were treated with 5F （0-80 lag/ml） for different time periods. Cytotoxicity was examined using a Ml-I- method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay （ELISA） and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species （ROS） level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results： 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor （AIF）, and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion： 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis.

Effects of Thermal and Hydric Conditions on Egg Incubation and Hatchling Phenotypes in Two Phrynocephalus Lizards

Flexible-shelled eggs of the lizards Phrynocephalus przewalskii and P. versicolor were incubated under different thermal and hydric conditions to elicit the effects of incubation environment on hatching success, embryonic development and duration as well as hatchling phenotypes. Embryogenesis of the two species was not sensitive to changes in the hydric environment except P. przewalskii incubated in 30°C group. Temperature significantly altered the duration of embryogenesis, with cooler temperatures leading to a longer incubation period. Hatching success was greater at 26 and 30°C than at 34°C. The hatchlings incubated at 26 and 30°C had longer snout-vent length, larger body mass, and better locomotor performance than those incubated at 34°C. Compared to P. przewalskii, P. versicolor had a shorter incubation period and yielded smaller hatchlings, which then had a higher survival rate in cooler and drier habitats. We conclude that an incubation temperature of 30°C would produce the best balance among developmental rate, hatching success, and post-hatching performance. We speculate that the upper temperature limit for incubation of P. versicolor eggs may be slightly higher than 34°C.

Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

Function of Lactate Dehydrogenase in Cardiac and Skeletal Muscle of Phrynocephalus Lizard in Relation to High-Altitude Adaptation

Poikilothermic animals living in high-altitude environments can be greatly affected by the anaerobic metabolism and lactate recycling, which are catalyzed by an enzyme called lactate dehydrogenase(LDH). However, the function and possible regulatory mechanisms of their anaerobic glycolysis remained elusive. We compared the difference in LDH between a native high-altitude(4 353 m) lizard, Phrynocephalus erythrurus, and a closely related species, Phrynocephalus przewalskii that lives in intermediate altitude environment(1 400 m). The activity of LDH, the concentration of lactate, the distribution of isoenzyme, and the mRNA amounts of Ldh-A and Ldh-B were determined. In cardiac muscle, the lactate-forming activity of P. erythrurus in LDH was higher than of P. przewalskii LDH at all three temperatures tested(10 °C, 25 °C and 35 °C), while lactate-oxidation activity of LDH was significantly different between the two species only at 25 °C and 35 °C. In skeletal muscle, both lactate-forming and lactate-oxidation rates of P. erythrurus were lower than that of P. przewalskii. There was a higher proportion of H subunit and a significantly higher expression of Ldh-B, with a concomitant decrease of lactate concentration in P. erythrurus. These results indicate that P. erythrurus may have a strong potential for anaerobic metabolism, which is likely adapted to the hypoxic environment at high altitudes. Furthermore, P. erythrurus is capable of oxidizing more lactate than P. przewalskii. The Ldh-A cDNA of the two species consists of a 999 bp open reading frame(ORF), which encodes 332 amino acids, while Ldh-B cDNA consists of a 1 002 bp ORF encoding 333 amino acids. LDHA has the same amino acid sequence between the two species, but three amino acid substitutions(V12 I, N21S and N318K) were observed in LDHB. Structure analysis of LDH indicated that the substitutions of residues Val12 and Asp21 in P. erythrurus could be responsible for the highaltitude adaptation. The LDH characteristics of LDH in P. erythrurus suggest unique adaptation strategies of anaerobic metabolism in hypoxia and cold environments at high altitudes for poikilothermic animals.

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

The inhibitory effects of NKX3.1 on IGF- 1R expression and its signalling pathway in human prostatic carcinoma PC3 cells

NKX3.1, which is a prostate-specific homeobox gene, plays an important role in prostate cancer and usually functions as a tumour suppressor gene. In this study, we investigated the inhibitory effect of NKX3,1 on insulin-like growth factor （IGF）- 1R expression and its downstream signalling pathway in PC3 cells, PC3 cells were stably transfected with NKX3.1 expression plasmid （pcDNA3.1-NKX3.1） or vector plasmid （pcDNA3.1＋）. The IGF-IR mRNA and protein expression levels were assessed in PC3-NKX3.1 transfectants by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blotting. The expression and activation of IGF-1/IGF-1R downstream signalling targets were examined by Western blotting and luciferase reporter assay. The cells were subsequently treated with relevant concentrations of IGF-1. The effect of IGF-1 on cell growth was examined by 3-（4,5）-dimethylthiahiazo（-z-yl）-3, 5-diphenytetrazoliumromide （MTT） assay and flow cytometry analysis. A significant suppression of IGF-1R mRNA and protein expression was observed after forced expression of NKX3.1 in PC3 cells. Correspondingly, the forced expression of NKX3.1 decreased IGF-l-induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERK1/2） and protein kinase B （AKT） and activation of the Elk-1 transcription factor and downregulated the expression of the downstream target genes c-fos and cyclin D1. Furthermore, the forced expression of NKX3.1 inhibited IGF-l-induced cell growth. In conclusion, NKX3.1 could downregulate IGF-1R expression and could inhibit IGF-1R-mediated mitogen-activated protein kinase （MAPK）/ERK and AKT signalling pathways, which might partially leads to the inhibition of IGF-l-induced cell growth. This study provides new insights into the molecular mechanisms that NKX3,1 exerts against prostate cancer and ultimately expands the scope of alternative approaches in advanced prostate cancer therapy.

Characterisation and Expression Analysis of Sox9 in the Multiocellated Racerunner, Eremias multiocellata

Sox9 is an important member of Sox family which is involved in a variety of developmental processes including sex determination and gonadal differentiation. The cDNA of Sox9 from multiocellated racerunner E. multiocellata was cloned using reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE）. The sequence contains a 1497 bp open reading frame, which encodes a 498 amino acid protein with a predicted molecular weight of 55.45 kDa. EmSox9 displays high similarity to those of reptiles, and shows an overall amino acid identity of 〉82%. We also investigated the tissue-specific expression of EmSox9 mRNA by realtime quantitative PCR. Sox9 mRNA is present in brain, heart, liver, kidney, gonads and muscle tissues of adult E. multiocellata, with the highest expression in brain and testis. The results indicate that Sox9 may play important roles in some tissues during E. multiocellata neural and gonadal development.

Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway

AIM:To investigate the mechanism by which galangin,a polyphenolic compound derived from medicinal herbs,induces apoptosis of hepatocellular carcinoma(HCC) cells.METHODS:The 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to measure cell viability.Apoptosis was evaluated by in situ uptake of propidium iodide and Hoechst 33258 and was then detected by fluorescence microscopy.Protein expressions were detected by Western blotting.To confirm the apoptotic pathway mediated by galangin,cells were transfected by bcl-2 gene to overexpress Bcl-2 or siRNA to down-regulate Bcl-2 expression.RESULTS:Galangin(46.25-370.0 μmol/L) exerted an anti-proliferative effect,induced apoptosis,and decreased mitochondrial membrane potential in a dose and time-dependent manner.Treatment with galangin induced apoptosis by translocating the pro-apoptotic protein Bax to the mitochondria,which released apoptosis-inducing factor and cytochrome c into the cytosol.Overexpression of Bcl-2 attenuated galangin-induced HepG2 cell apoptosis,while decreasing Bcl-2 expression enhanced galangin-induced cell apoptosis.CONCLUSION:Our data suggests that galangin mediates apoptosis through a mitochondrial pathway,and may be a potential chemotherapeutic drug for the treatment of HCC.

QSAR Studies on the Toxicity of Nitrobenzenes to Population Growth of Tetrahymena Pyriformis

The IGC 50 (50% inhibitory growth concentration) values of 27 nitrobenzenes were determined for the population growth endpoint of Tetrahymena pyriformis . The toxicity order of the observed compounds was found to be as follows: dinitro compounds>mononitro compounds, dichloronitrobenzenes>monochloronitrobenzenes, meta substituted nitrobenzenes>ortho /para substituted nitrobenzenes(NT, NPh, NAnis) except for the dinitrobenzenes and nitroanilines(DNB, NAn). Quantitative structure activity relationships(QSARs) were developed with the logarithm of the reciprocal of IGC 50 [lg(IGC 50 ) -1 ] in mole liter as dependent variable and six molecular descriptors lg P , 1 X Ⅴ, I, 1K a, ∑σ - and E LUMO as independent variables. Via multiple regression analysis, one best equation was obtained: lg(IGC 50 ) -1 =3.029+0.860∑ σ -+0.341I n=27, r=0.924, r 2=0.854, s=0.265, f=70.44 , Pr> f =0.000 1 The equation was used to estimate IGC 50 for five analogues.

Downregulation of thioredoxin reductase 1 expression in the substantia nigra pars compacta of Parkinson's disease mice

Because neurons are susceptible to oxidative damage and thioredoxin reductase 1 is extensively distributed in the central nervous system and has antioxidant properties, we speculated that the enzyme may be involved in the pathogenesis of Parkinson＇s disease. A Parkinson＇s disease model was produced by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into C57BL/6 mice. Real-time reverse transcription-PCR, western blot analysis and colorimetric assay showed that the levels of thioredoxin reductase 1 mRNA and protein were decreased, along with a significant reduction in thioredoxin reductase activity, in the midbrain of Parkinson＇s disease mice compared with normal mice. Immunohistochemical staining revealed that the number of thioredoxin reductase 1-positive neurons in the substantia nigra pars compacta of Parkinson＇s disease mice was significantly decreased compared with normal mice. These experimental findings suggest that the expression of thioredoxin reductase 1 in the substantia nigra pars compacta of Parkinson＇s disease mice is significantly decreased, and that the enzyme may be associated with disease onset.

Heme oxygenase-1 system and gastrointestinal inflammation:A short review

Heme oxygenase-1(HO-1) system catalyzes heme to biologically active products:carbon monoxide,biliverdin/bilirubin and free iron.It is involved in maintaining cellular homeostasis and many physiological and pathophysiological processes.A growing body of evidence indicates that HO-1 activation may play an important protective role in acute and chronic inflammation of gastrointestinal tract.This review focuses on the current understanding of the physiological significance of HO-1 induction and its possible roles in the gastrointestinal inflammation studied to date.The ability to upregulate HO-1 by pharmacological means or using gene therapy may offer therapeutic strategies for gastrointestinal inflammation in the future.

From basic researches to new achievements in therapeutic strategies of KRAS-driven cancers

Among the numerous oncogenes involved in human cancers, KRAS represents the most studied and best characterized cancerrelated genes.Several therapeutic strategies targeting oncogenic KRAS(KRASonc) signaling pathways have been suggested,including the inhibition of synthetic lethal interactions, direct inhibition of KRASonc itself, blockade of downstream KRASonc effectors, prevention of post-translational KRASonc modifications, inhibition of the induced stem cell-like program, targeting of metabolic peculiarities, stimulation of the immune system, inhibition of inflammation, blockade of upstream signaling pathways,targeted RNA replacement, and oncogene-induced senescence.Despite intensive and continuous efforts, KRASonc remains an elusive target for cancer therapy.To highlight the progress to date, this review covers a collection of studies on therapeutic strategies for KRAS published from 1995 to date.An overview of the path of progress from earlier to more recent insights highlight novel opportunities for clinical development towards KRASonc-signaling targeted therapeutics.

Adult neural stem cell dysfunction in the subventricular zone of the lateral ventricle leads to diabetic olfactory defects

Sensitive smell discrimination is based on structural plasticity of the olfactory bulb,which depends on migration and integration of newborn neurons from the subventricular zone.In this study,we examined the relationship between neural stem cell status in the subventricular zone and olfactory function in rats with diabetes mellitus.Streptozotocin was injected through the femoral vein to induce type 1 diabetes mellitus in Sprague-Dawley rats.Two months after injection,olfactory sensitivity was decreased in diabetic rats.Meanwhile,the number of Brd U-positive and Brd U＋/DCX＋double-labeled cells was lower in the subventricular zone of diabetic rats compared with agematched normal rats.Western blot results revealed downregulated expression of insulin receptorβ,phosphorylated glycogen synthase kinase 3β,and β-catenin in the subventricular zone of diabetic rats.Altogether,these results indicate that diabetes mellitus causes insulin deficiency,which negatively regulates glycogen synthase kinase 3β and enhances β-catenin degradation,with these changes inhibiting neural stem cell proliferation.Further,these signaling pathways affect proliferation and differentiation of neural stem cells in the subventricular zone.Dysfunction of subventricular zone neural stem cells causes a decline in olfactory bulb structural plasticity and impairs olfactory sensitivity in diabetic rats.

The miRNA let-7al inhibits the expression of insulin-like growth factor 1 receptor （IGFIR） in prostate cancer PC-3 cells

Reduced microRNA （miRNA） let-7a expression and the activation of insulin-like growth factor-1 receptor （IGFIR） signalling are both involved in prostate cancer and progression. In the present study, we demonstrated that the growth inhibitory effect of let-7al is directly related to targeting IGFIRgene expression in PC-3 cells. TargetScan predicted three potential target sites （T1, T2 and T3） of let-7a in the 3＇ untranslational region （3＇ UTR） of IGFIR mRNA. Real-time PCR, Western blot and luciferase reporter assays were used to detect the effects of let-7al overexpression or let-7al inhibitor on the IGFIRgene expression in PC-3 cells. The results indicated that let-7al could inhibit IGFIR expression by directly targeting the T1 and T2 sites in the 3＇ UTR of the IGFIR mRNA. We then used RT-PCR, luciferase reporter assays, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-2H-tetrazolium bromide （MTT） assay, flow cytometry and Hoechst 33342 staining to examine whether let-7al-mediated inhibition of IGFIR expression also affects the IGFIR-mediated signalling events, including Elk1 activity and c-fos gene expression, proliferation, apoptosis and cell cycle. We demonstrated that let-7al-mediated IGFIR downregulation was accompanied by attenuation of Elk1 activity and c-fos expression, inhibition of cell proliferation, enhanced apoptosis and cell cycle arrest, and that loss function of let-7al via inhibition can upregulate IGF1R accompanied by an increase of Elk1 activity and c-fos expression, thereby enhancing cell proliferation. Altogether, these findings sueeest that let-7a mav be novel therapeutic candidate for Drostate cancer.

Inhalation of shin-I essential oil enhances lactate clearance in treadmill exercise

Objective:To evaluate the effect of Shin-I essential oil inhalation on blood lactate changes in rats subjected to treadmill exercise.Methods:Adult male Sprague Dawley rats(n=12) were randomly divided into the control or the Shin—1 group.Rats were subjected to a treadmill exercise program(15 m/min for 30 mim.After exercise,rats were exposed to 200 ui.of water or Shin—I essential oil.res|ieclively.using a nebulizer for 180 min during the recovery period.Blood samples were collected every 15 min.Blood glucose and lactate concentrations were determined in a CMA 600 analyzer.Results:The basal glucose and lactate levels wen? no significantly different between two groups.After exercise,glucose levels were slightly increased to about 110%-120%of the basal level in both groups.lactate levels of both groups reached to 110%-140%of basal levels during exercise.In the recovery period,lactate levels further increased to 180%of the basal level and were maintained at a plateau in the control group.However,lactate levels gradually decreased to 609—657 of the basal level in the Shin-I group.Lactate clearance was significantly enhanceil after Shin-I essential oil inhalation.Conclusions:Our results provide evidence that Shin-I essential oil inhalation may accelerate recovery after exercise in rats.

Effect of clausenamide on hippocampal neuron apoptosis induced by sodium nitroprusside

BACKGROUND： Aggregation of β-amyloid peptide （A β ）, excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease （AD）. （ - ） clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free radicals, resisting A β neurotoxicity and nerve cell apoptosis, inhibiting over phosphorylation of tau protein, and improving central cholinergic system. However, whether （-） clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE： To observe the effect of （ - ） clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside （SNP） and analyze the possible pathways. DESIGN： Contrast observation. SETTING： Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS： A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synthesized by Beijing Huada Genetic Engineering Company and （-） clausenamide （99.6%） was provided by Pharmacological Department of Chinese Academy of Medical Sciences. SNP was provided by Sigma Company. METHODS： Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 la mol/L （ - ） clausenamide for 6 hours; the culture medium was gotten rid of and neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above mentioned volume of （ - ） clausenamide and 50 μ mol/L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group （culture medium control）, model group （SNP treatment） and experimental group [（ - ） clausenamide ＋ SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chromatometry; levels of mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction （RT-PCR）; expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES： ① Effect of （-） clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ②bcl-2 and bax mRNA and protein expression ofhippocampal neurons. RESULTS： ①Survival rate of hippocampal neurons： Survival rate of hippocampal neurons affected by 0.4 - 1.6 μ mol/L （ - ） clausenamide was higher in the experimental group than the model group （P 〈 0.01）, and the survival rate was increased with the larger volume of （ - ） clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μ mol/L, and it had obvious dosage dependence （P 〈 0.01）. ②Expression of bcl-2 and bax mRNA： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③Expression of Bcl-2 and Bax protein： Hippocampal neurons were pretreated with 0.2 - 1.6 μ mol/L （ - ） clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually. This suggested that, with the increase of concentration, expression of Bcl-2 protein was increased simultaneously. However, when strap of PCR product of Bax protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION： （ - ） clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression of pro-apoptotic bax gene.

Carbohydrate-associated epitope-based anti-cancer drugs and vaccines

RP215 is one of the three thousand monoclonal antibodies (Mabs) which were generated against the OC-3-VGH ovarian cancer cell line. RP215 was shown to react with a carbohydrate-associated epitope located specifically on glycoproteins, known as CA215, from cancer cells. Further molecular analysis by matrix adsorption laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) revealed that CA215 consists mainly of immunoglobulin super-family (IgSF) proteins, including immunoglobulins, T-cell receptors, and cell adhesion molecules, as well as several other unrelated proteins. Peptide mappings and glycoanalysis were performed with CA215 and revealed high-mannose and complex bisecting structures with terminal sialic acid in N-glycans. As many as ten O-glycans, which are structurally similar to those of mucins, were also identified. In addition, two additional O-linked glycans were exclusively detected in cancerous immunoglobulins but not in normal B cell-derived immunoglobulins. Immunizations of mice with purified CA215 resulted in the predominant generation of RP215-related Mabs, indicating the immunodominance of this carbohydrate-associated epitope. Anti-idiotype (anti-id) Mabs of RP215, which were generated in the rat, were shown to contain the internal images of the carbohydrate-associated epitope. Following immunizations of these anti-id Mabs in mice, the resulting anti-anti-id (Ab3) responses in mice were found to be immunologically similar to that of RP215. Judging from these observations, anti-id Mabs, which carry the internal image of the RP215-specific epitope, may be suitable candidates for anticancer vaccine development in humans.

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6 ST(p.R281 Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6 st p.R279 Q. We show that human GP130 p.R281 Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6 st p.R279 Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11 RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.