Synthesis and Evaluations of Novel Apocynin Derivatives as Anti-Glioma Agents.

Apocynin (4-hydroxy-3-methoxyacetophenone) is a natural polyphenolic compound with multiple biological activities. In the present study, a series of apocynin derivatives were designed and synthesized. The in silico ADMET prediction, blood-brain barrier (BBB) penetration assay, anti-NADPH oxidase activity, reactive oxygen species (ROS) levels, and anti-glioma effects of these apocynin derivatives were evaluated. The anti-glioma mechanisms of candidate compounds were studied by flow cytometer and Western blot. The results showed that D31 exhibited higher BBB penetration, increased ROS generations and significant anti-glioma effects both in vitro and in vivo. Further studies showed that D31 inhibited the activations of NF-κB pathway. Overall, our data demonstrated that D31 inhibited growth and induced apoptosis of glioma, which might be caused by ROS-related NF-κB activation. The current study suggested that D31 could be further explored for its potential use in anti-glioma therapy.

Role of catalpol in ameliorating the pathogenesis of experimental autoimmune encephalomyelitis by increasing the level of noradrenaline in the locus coeruleus.

The endogenous neurotransmitter, noradrenaline, exerts anti-inflammatory and neuroprotective effects in vivo and in vitro. Reduced noradrenaline levels results in increased inflammation and neuronal damage. The primary source of noradrenaline in the central nervous system is tyrosine hydroxylase (TH)­positive neurons, located in the locus coeruleus (LC). TH is the rate­limiting enzyme for noradrenaline synthesis; therefore, regulation of TH protein expression and intrinsic enzyme activity represents the central means for controlling the synthesis of noradrenaline. Catalpol is an iridoid glycoside purified from Rehmannia glutinosa Libosch, which exerts a neuroprotective effect in multiple sclerosis (MS). The present study used an experimental mouse model of autoimmune encephalomyelitis to verify the neuroprotective effects of catalpol. Significant improvements in the clinical scores were observed in catalpol­treated mice. Furthermore, catalpol increased TH expression and increased noradrenaline levels in the spinal cord. In primary cultures, catalpol exerted a neuroprotective effect in rat LC neurons by increasing the noradrenaline output. These results suggested that drugs targeting LC survival and function, including catalpol, may be able to benefit patients with MS.

Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance, Ca2+-Activated K+ Channels In Vitro.

Increasing evidence suggests that low to moderate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca2+-activated K+ channel (BKCa) α-subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) compared with controls. Preconditioning with low-dose ethanol (10 mmol/L) increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca2+ levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol preconditioning was antagonized by a BKCa channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BKCa channel by 10 mmol/L ethanol. The above results indicated that low-dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca2+ and preventing neuronal apoptosis, and this is mediated by BKCa channel activation.

Reactivated astrocytes as a possible source of oligodendrocyte precursors for remyelination in remitting phase of experimental autoimmune encephalomyelitis rats.

Multiple sclerosis (MS) is ademyelinating disease in the central nervous system (CNS). Majority of the MS patients show relapsing-remitting disease course. Evidences show that oligodendrocyte precursor cells (OPCs), which remain relatively quiescent in normal adult CNS, play a key role in the remitting phase by proliferation and remyelination. In the present study, we found that spinal cord astrocytesco-expressed progenitor cell marker and oligodendroglial lineage markers in the remittance phase in adult rat experimental autoimmune encephalomyelitis (EAE) model. We suggest that activated astrocyte could de-differentiate into OPCs and re-differentiate into mature oligodendrocytes, raising the possibility that astrocytes can be a potential source of OPCs in the adult demyelinated spinal cord.

Imatinib mesylate therapy for patients with chronic myeloid leukemia:long-term out-come from a single center in China / 中国肿瘤临床

Objective:Imatinib is extensively used as a first-line therapeutic agent for patients with chronic myeloid leukemia (CML) at the chronic phase (CP). Although CML patients undergoing imatinib treatment are enrolled mainly in the Glivec International Patient Assistance Program (GIPAP) in China since 2003, limited data have been reported on the long-term outcome of these patients. This study aims to compare the treatment response and prognosis of CML-CP patients who received different treatments from January 2003 to December 2013 in the First Affiliated Hospital of Nanchang University. Methods:A total of 295 patients were enrolled, includ-ing 185, 30, 50, and 30 patients for imatinib, interferon-alpha (IFN-α) plus Ara-C, hydroxycarbamide (HU), or allogeneic hematopoietic stem cell transplantation (Allo-HSCT) treatments, respectively. Results:Patients in imatinib and Allo-HSCT groups achieved excellent complete hematologic remission (CHR) (i.e., 96.7%vs. 96.7%), complete cytogenetic response (CCyR) (i.e., 89.7%vs. 93.3%), and com-plete molecular remission (CMoR) (i.e., 49.7%vs. 83.3%, P=0.001). However, significantly low rates of CHR, CCyR, McyR, and CMoR were observed in IFN-αand HU groups. Moreover, patients from imatinib group showed longer overall survival (OS) time than patients from other groups (P<0.001), even patients in Allo-HSCT group (10-year OS, 89.0%vs. 67.0%, P<0.001) because of high risk of Allo-HSCT-related complication. Multivariate analysis showed that receiving imatinib treatment (HR=5.267, 95%CI:1.054-1.940, P=0.022) and achieving CCyR (HR=9.541, 95%CI:1.692-10.513, P=0.002) were independent predictors for OS. Conclusion:Imatinib treatment may be an optimal first-line choice for Chinese patients with CML-CP who have not received any previous treatments.

Catalpol induces oligodendrocyte precursor cell-mediated remyelination in vitro.

In demyelinating diseases such as multiple sclerosis, one of the treatment strategies includes remyelination using oligodendrocyte precursor cells (OPC). Catalpol, the extract of radix rehmanniae, is neuroprotective. Using an OPC culture model, we showed that 10 µM catalpol promotes OPC proliferation, cell migration and differentiation into mature oligodendrocytes. The 10 µM catalpol displayed stronger effects on OPCs migration and oligodendrocyte differentiation. These results suggest that catalpol has a potential role in promoting remyelination in demyelinating diseases, and is of therapeutic interest.

Inhibitory Effect of Curcumin on Proliferation of CD34(+) Acute Myeloid Leukemia Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effect of curcumin on proliferation of CD34(+) acute myeloid leukemia cells and its mechamism.</p><p><b>METHODS</b>KG1a and Kasumi-1cell lines were treated with curcumin of different concentrations (0, 40, 60, 80 µmol/L). The effect of curcumin on cell viability and proliferation was detected by trypan blue staining and cell count. The effect of curcumin on distribution of NF-κB P65 subunit was analyzed by immunofluorescence and Western blot.</p><p><b>RESULTS</b>The curcumin inhibited proliferation of KG1a and Kasumi-1 cells in a dose-dependent manner. Western blotting showed that curcumin led to significant down-regulation of NF-κB P65 nuclear protein expression. Immunofluorescence assay showed that treatment with 40 µmol/L of curcumin for 48h suppressed the nuclear translocation of NF-κB p65 in KG1a and Kasumi-1 cells.</p><p><b>CONCLUSION</b>The curcumin suppresses cell growth of KG1a and Kasumi-1 cells, its mechanism may be related to inhibitory effect of curcumin on NF-κB p65 nucleus protein.</p>

Incidence of JAK2V617F mutation in myeloproliferative diseases and its clinical significance / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the incidence of JAK2V617F gene point mutation in patients with myeloproliferatives diseases (MPD) and its clinical significance.</p><p><b>METHODS</b>Genomic DNA from bone marrow and peripheral blood cells were extracted from 68 patients with MPD. Allele specific polymerase chain reaction was used to amplify the exon 12 of JAK2 gene which harbours V617F mutation. The PCR products were identified by DNA sequencing. JAK2V617F gene point mutation and its impact on peripheral blood cells were analyzed.</p><p><b>RESULTS</b>The incidence of JAK2V617F mutation in 68 patients with MPD was 65.28 %. The positive rate of JAK2V617F point mutation was 77.77 % in patients with PV (36/59), 56.52 % in patients with ET (23/59) and 44.44 % in patients with IMF (4/9). In all groups, the incidence of JAK2V617F point mutation in bone marrow and peripheral blood were equal. Patients with JAK2V617F mutation in PV group had higher counts of white blood cell and hemoglobin in peripheral blood than patients without JAK2V617F point mutation (P <0.05). Patients with JAK2V617F mutation in ET group had higher counts of white blood cell than those without JAK2V617F mutation (P <0.05); there was no significant difference in platelet count.</p><p><b>CONCLUSION</b>JAK2V617F point mutation can affect the hematologic features, which may be of diagnostic value for MDP with negative BCR-ABL gene.</p>

Combined effect of TNF-related apoptosis induced ligand and Ara-C in inducing apoptosis of HL-60 cells and its mechanism / 中国实验血液学杂志

To investigate the combined effect of human recombinant soluble TNF-related apoptosis induced ligand (hrsTRAIL) with Ara-C or alone on HL-60 leukemia cell lines and its mechanism, human leukemia cell lines HL-60 were cultured in vitro. HL-60 cells were divided into 5 groups: control group, Ara-C group, rsTRAIL group, Ara-C + rsTRAIL simultaneously given group, Ara-C + rsTRAIL tandem given group (Ara-C followed by rsTRAIL group). The cytotoxic effect was measured by MTT assay; cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining; the expression level of DR5 on surface of HL-60 cells treated with Ara-C at different concentrations for 24 hours was determined by flow cytometry. The expression level of DR5 on surface of HL-60 cells and caspase-8 activity in HL-60 cells of rsTRAIL group and Ara-C + rsTRAIL tandem group was determined by flow cytometry. The result showed that rsTRAIL could inhibit the proliferation of HL-60 cells and induce apoptosis of HL-60 cells in a concentration-dependent manner. The apoptosis rate of HL-60 cells in Ara-C + rsTRAIL tandem given group was higher than that in Ara-C + rsTRAIL simultaneously given group, the expression level of DR5 on surface of HL-60 cells and intracellular activity of caspase-8 in Ara-C + rsTRAIL tandem given group were higher than those in rsTRAIL group. When HL-60 cells treated with 5 and 10 mg/L of Ara-C for 24 hours, the expression level of DR5 on surface of HL-60 cells was higher than that in control group. It is concluded that rsTRAIL can inhibit the proliferation of HL-60 cells, and induce apoptosis of HL-60 cells. Ara-C can upregulate DR5 expression on the surface of HL-60 cells and enhance the effect of rsTRAIL-inducing apoptosis. Tandem treatment of HL-60 cells with Ara-C followed by rsTRAIL induce more apoptosis than that of co-treatment with rsTRAIL and Ara-C. Ara-C and rsTRAIL has a synergistic inhibitory effect on growth of HL-60 cells. The mechanism may correlate with up-regulation of the expression level of DR5 and/or caspase-8 in HL-60 cells by Ara-C.

High efficiency of L-glutamine production by coupling genetic engineered bacterial glutamine synthetase with yeast alcoholic fermentation system / 生物工程学报

Glutamine is an important conditionally necessary amino acid in human body. The effort is to establish a new and high efficient L-glutamine production system instead of traditional fermentaion. In this paper, high efficiency of L-glutamine production is obtained by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system. Glutamine Synthetase gene (glnA) was amplified from Bacillus subtilis genomic DNA with primers designed according to sequences reported in EMBL data bank, then it was inserted into expression vector PET28b, the sequence of glnA was proved to be the same as that reported in the data bank by DNA sequencing. After transformation of this recombinant plasmid PET28b-glnA into BL-21 (DE3) strain, Lactose and IPTG were used to induce GS expression at 37 degrees C separately. Both of them can induce GS expression efficiently. The induced protein is proved to be soluble and occupies about 80% of the total proteins by SDS-PAGE analysis. The soluble GS was purified by Ni2+ chelating sepharose colum. After purification, the purified enzyme was proved active. Results reveal that the optmum temperature of this enzyme is 60 degrees C and optmum pH is 6.5 in biosynthetic reaction by using glutamate, ammonium choloride and ATP as substrates. After induction, the enzyme activity in crude extract of BL-21/PET28b-glnA is 83 times higher than that of original BL-21 extract. Mn2+ can obviously increase the activity and stability of this enzyme. Experiments show that the transformation efficiency of glutamate to glutamine is more than 95%. Because of the high cost from ATP, a system coupling GS with yeast for ATP regenaration was established. In this system, GS utilizes ATP released by yeast fermentation to synthesize L-glutamine. Yeast was treated by 2% toluence to increase its permeability and a yeast named YC001 with high yield of glutamine by coupling with recombinant GS was obtained. The good efficiency was achieved with the presence of 250 mmol/L glucose and 200 mmol/L phosphate, the transformation efficiency of glutamate to glutamine in this system is more than 80%, the average yield of glutamine is about 22g/L. This provides the basis for future large scale production of L-glutamine.

Survival and differentiation of transplanted neural stem cells in mice brain with MPTP-induced Parkinson disease.

AIM: To determine survival and differentiation of cultured neural stem cells (NSCs) into viable and functional neurons upon transplantation into mice brain of MPTP-induced Parkinson disease (PD). METHODS: Mouse model of PD was established with two subcutaneous (sc) injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 40 mg/kg) twice, 16 h apart. NSCs isolated from rat embryo midbrain were cultured in clonal density. After labeled with 5-bromo-2'-deoxyuridine (BrdU), the NSCs were transplanted into the uni- or bi-lateral striatum of PD mouse. Tyrosine hydroxylase (TH) immunofluorescence was used to evaluate the toxicity of MPTP on the neural cells in the substantia nigra. Immunohistology and laser confocal microscope were used to detect the survival and differentiation of transplanted NSCs. RESULTS: The cultured NSCs generated neurospheres and differentiated into neuron and astrocyte. It indicated that the cultured NSCs were multipotent and self-renewal in vitro. TH-positive neural cells were significantly reduced in the substantia nigra. Immunohistology showed that the uni- or bi-lateral transplanted NSCs survived in the brain of PD model mouse. Laser confocal microscope indicated that some transplanted NSCs could properly differentiate into targeted TH-positive neural cells in vivo. CONCLUSION: The transplanted multipotent NSCs could survive and differentiate into functional dopamine neurons.