Effects of a unilateral stereotaxic injection of Tinuvin 123 into the substantia nigra on the nigrostriatal dopaminergic pathway in the rat

Tinuvin 123, a compound used in the manufacture of plastics, has recently been suggested to possibly cause Parkinson's disease (PD). Herein, we revisited this issue by assessing the effect of Tinuvin 123 on dopaminergic neurons of the substantia nigra following its stereotaxic injection in the rat. Twenty-one days post unilateral stereotaxic injection of Tinuvin 123, systemic injection of both apomorphine and amphetamine caused rotations toward the side of the lesion in these rats. Tinuvin 123 produced a small to moderate dose-dependent reduction in striatal levels of dopamine and metabolites on the side of the lesion. This compound also produced dramatic cell loss in the substantia nigra on the side of the lesion. However, the loss of cells lacked the phenotypic specificity for tyrosine hydroxylase (TH)-positive neurons that is expected with a dopaminergic neurotoxin. Indeed, aside from a robust glial reaction, both TH-positive and glutamic acid dehydrogenase (GAD)-positive neurons were destroyed, and near the site of the injection, there was complete tissue destruction. This study indicates that, using this mode of injection, Tinuvin 123 exerts a dramatic tissue toxicity without any evidence of specificity for dopaminergic neurons. Thus, our data argues against a role for Tinuvin 123 as an environmental toxin causing a clinical condition characterized by the selective loss of dopaminergic neurons as seen in PD.     

Relation between myocardial response to dobutamine stress and sympathetic nerve activation in patients with idiopathic dilated cardiomyopathy: A comparison of123I-MIBG scintigraphic and echocardiographic data

It is likely that a close association exists between findings obtained by two methods: dobutamine stress echocardiography and 123I-MIBG scintigraphy. Both of these methods are associated with beta-adrenergic receptor mechanisms. This study was conducted to demonstrate the relation between myocardial response to dobutamine stress and sympathetic nerve release of norepinephrine in the failing heart. In 12 patients with heart failure due to idiopathic dilated cardiomyopathy, the myocardial effects of dobutamine stress were evaluated by low-dose dobutamine stress echocardiography: and sympathetic nerve function was evaluated by scintigraphic imaging with iodine-123 [123I] meta-iodobenzylguanidine (MIBG), an analogue of norepinephrine. Echocardiography provided quantitative assessment of wall motion and left ventricular dilation; radiotracer studies with 123I-MIBG provided quantitative assessment of the heart-to-mediastinum (H/M) uptake ratio and washout rate. Results showed that H/M correlated with baseline wall motion (r = 0.682, p = 0.0146), wall motion after dobutamine stress (r = 0.758, p = 0.0043), the change in wall motion (r = 0.667, p = 0.0178), and with left ventricular diastolic diameter (r = 0.837, p = 0.0007). In addition, the 123I-MIBG washout rate correlated with baseline wall motion (r = 0.608, p = 0.0360), wall motion after dobutamine stress (r = 0.703, p = 0.0107), and with the change in wall motion (r = 0.664, p = 0.0185). Wall motion, especially in the myocardial response to dobutamine stress, is related to sympathetic nerve activity in heart failure.     

Double-injection method for sequentially measuring cerebral blood flow with N-isopropyl-(123I)p-iodoamphetamine

We investigated the accuracy of a double-injection method for sequentially measuring cerebral blood flow (CBF) with N-isopropyl-(123I)p-iodoamphetamine (IMP) in simulation studies based on patient data and in clinical studies. The unidirectional clearance of IMP from the blood to the brain (K1; nearly equal to CBF) in the first and second sessions was calculated by means of a microsphere model. The K1 values in the first session (K1I) were calculated from Cb(5)/Int_CaI, where Cb(5) and Int_CaI are values for brain radioactivity 5 min after the first injection and for arterial blood radioactivity obtained by 5-min continuous sampling. The K1 values in the second session (K1II) were calculated by means of the following four methods. Method 1: [Cb(tz + 5) - Cb(tz)]/[Int_CaII - Ca(tz) x 5], where Cb(tz+5) and Cb(tz) are the brain radioactivity levels 5 min after the second injection and at the time the second session was started (tz), respectively. Int_CaII and Ca(tz) are the arterial blood radioactivity levels obtained by 5-min continuous sampling after the second injection and at tz, respectively. Method 2: [Cb(tz + 5) - Cb(tz)]/[Int_CaI x R], where R is the injection dose ratio. Method 3: [Cb(tz + 5) - Cb(tz) x exp(- K1I x 5/lambda)]/Int_CaII, where lambda is the population averaged partition coefficient. Method 4: same as Method 3 except that K1I was replaced by K1II obtained by means of Method 2. Theoretically, Method 4 appeared to be the best of the four methods. The change in K1 during the second session obtained by Method 1 or 2 largely depended on R and tz, whereas Method 3 or 4 yielded a more reliable estimate than Method 1 or 2, without largely depending on R and tz. Since Method 2 was somewhat superior to other methods in terms of noninvasiveness and simplicity, it also had the potential for routine clinical use. The reproducibility of two sequential measurements of K1 was investigated with clinical data obtained without any intervention. The response of CBF to acetazolamide challenge was also assessed by the above four methods. The knowledge gained by this study may assist in selecting a method for sequentially measuring CBF with a double injection of IMP.     

Radiolabeled Anti-CD45 Antibody with Reduced-Intensity Conditioning and Allogeneic Transplantation for Younger Patients with Advanced Acute Myeloid Leukemia or Myelodysplastic Syndrome

We treated patients under age 50 years with iodine-131 (¹³¹I)–anti-CD45 antibody combined with fludarabine and 2 Gy total body irradiation to create an improved hematopoietic cell transplantation (HCT) strategy for advanced acute myeloid leukemia or high-risk myelodysplastic syndrome patients. Fifteen patients received 332 to 1561 mCi of ¹³¹I, delivering an average of 27 Gy to bone marrow, 84 Gy to spleen, and 21 Gy to liver. Although a maximum dose of 28 Gy was delivered to the liver, no dose-limiting toxicity was observed. Marrow doses were arbitrarily capped at 43 Gy to avoid radiation-induced stromal damage; however, no graft failure or evidence of stromal damage was observed. Twelve patients (80%) developed grade II graft-versus-host disease (GVHD), 1 patient developed grade III GVHD, and no patients developed grade IV GVHD during the first 100 days after HCT. Of the 12 patients with chronic GVHD data, 10 developed chronic GVHD, generally involving the skin and mouth. Six patients (40%) are surviving after a median of 5.0 years (range, 4.2 to 8.3 years). The estimated survival at 1 year was 73% among the 15 treated patients. Eight patients relapsed, 7 of whom subsequently died. The median time to relapse among these 8 patients was 54 days (range, 26 to 1364 days). No cases of nonrelapse mortality were observed in the first year after transplantation. However, 2 patients died in remission from complications of chronic GVHD and cardiomyopathy, at 18 months and 14 months after transplantation, respectively. This study suggests that patients may tolerate myeloablative doses >28 Gy delivered to the liver using ¹³¹I-anti-CD45 antibody in addition to standard reduced-intensity conditioning. Moreover, the arbitrary limit of 43 Gy to the marrow may be unnecessarily conservative, and continued escalation of targeted radioimmunotherapy doses may be feasible to further reduce relapse.     

Nobiletin and its derivatives overcome multidrug resistance (MDR) in cancer: total synthesis and discovery of potent MDR reversal agents

Our recent studies demonstrated that the natural product nobiletin (NOB) served as a promising multidrug resistance (MDR) reversal agent and improved the effectiveness of cancer chemotherapy in vitro. However, low aqueous solubility and difficulty in total synthesis limited its application as a therapeutic agent. To tackle these challenges, NOB was synthesized in a high yield by a concise route of six steps and fourteen derivatives were synthesized with remarkable solubility and efficacy. All the compounds showed improved sensitivity to paclitaxel (PTX) in P-glycoprotein (P-gp) overexpressing MDR cancer cells. Among them, compound 29d exhibited water solubility 280-fold higher than NOB. A drug-resistance A549/T xenograft model showed that 29d, at a dose of 50 mg/kg co-administered with PTX (15 mg/kg), inhibited tumor growth more effective than NOB and remarkably increased PTX concentration in the tumors via P-gp inhibition. Moreover, Western blot experiments revealed that 29d inhibited expression of NRF2, phosphorylated ERK and AKT in MDR cancer cells, thus implying 29d of multiple mechanisms to reverse MDR in lung cancer.     

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells in vitro and in vivo

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells in vitro and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [¹²⁵I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.     

Effects of Hyperkinetic,a β Subunit of Shaker Voltage-Dependent K+ Channels,on the Oxidation State of Presynaptic Nerve Terminals

The Drosophila Hyperkinetic (Hk) gene encodes a β subunit of Shaker (Sh) K⁺ channels and shows high sequence homology to aldoketoreductase. Hk mutations are known to modify the voltage dependence and kinetics of Sh currents, which are also influenced by the oxidative state of the N-terminus region of the Sh channel, as demonstrated in heterologous expression experiments in frog oocytes. However, an in vivo role of Hk in cellular reduction/oxidation (redox) has not been demonstrated. By using a fluorescent indicator of reactive oxygen species (ROS), dihydrorhodamine-123 (DHR), we show that the presynaptic nerve terminal of larval motor axons is metabolically active, with more rapid accumulation of ROS in comparison with muscle cells. In Hk terminals, DHR fluorescence was greatly enhanced, indicating increased ROS levels. This observation implicates a role of the Hk β subunit in redox regulation in presynaptic terminals. This phenomenon was paralleled by the expected effects of the mutations affecting glutathione S-transferase S1 as well as applying H₂O₂ to wild-type synaptic terminals. Thus, our results also establish DHR as a useful tool for detecting ROS levels in the Drosophila neuromuscular junction.     

Phagocytosis of co-developing neutrophil progenitors by dendritic cells in a culture of human CD34<Superscript>+</Superscript> cells with granulocyte colony-stimulating factor and tumor necrosis factor-α

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the differentiation of CD34(+) cells toward dendritic cells (DCs). We have previously shown that DCs are co-generated from human CD34(+) cells during erythroid or megakaryocytic differentiation in the presence of TNF-alpha, and those DCs are able to stimulate autologous T cell proliferation. The aim of this study was to learn whether the co-stimulation of granulocyte colony-stimulating factor (G-CSF) and TNF-alpha would generate neutrophil progenitors and DCs together from human CD34(+) cells, and if this was the case, to clarify the phenotypic and functional characteristics of these DCs. When highly purified human CD34(+) cells were cultured for 7 days with G-CSF alone, the generated cells predominantly expressed a granulocyte marker, CD15, and then differentiated into neutrophils after 14 days of culture. The addition of TNF-alpha with G-CSF markedly decreased the number of CD15(+) cells without affecting the total number of cells during 7 days of culture. Almost one third of the generated cells were positive for CD11c and CD123. Furthermore, CD11c(+) cells were found to phagocytose CD15(+) cells and were able to induce allogeneic, but not autologous, T cell proliferation in the mixed lymphocyte reaction (MLR). On the other hand, the CD11c(+) cells generated by TNF-alpha and cytokines capable of inducing erythroid differentiation were able to stimulate autologous T cells. There was a difference in the expression of CD80, CD83 and CD86 among CD11c(+) cells induced by G-CSF plus TNF-alpha and those generated by interleukin-3, stem cell factor, and erythropoietin plus TNF-alpha. These results indicate that the co-stimulation of human CD34(+) cells with G-CSF and TNF-alpha induces the phagocytosis of co-developing neutrophil progenitors by DCs, and the stimulatory effects of these DCs on autologous T cells is different from that of DCs generated from CD34(+) cells during erythroid differentiation.     

内皮素-1对大鼠离体心脏缺血再灌注损伤的保护作用

目的观察内皮素-1(ET-1)对大鼠离体心脏缺血再灌注损伤的影响以及不同浓度ET-1的不同效应。方法健康雄性SD大鼠30只,体重250~300 g,建立Langendorff离体心脏灌注模型。随机分为5组:对照组、ET-1组(0.1、1、10 nmol/L)和BQ-123组(2μmol/L),每组6只。所有离体心脏均平衡20 min,全心缺血30 min,再灌注60 min。观察各组在缺血再灌注损伤后各时间点的左室发展压(LVDP)、左室舒张末压(LVEDP)、冠脉流量(CF)以及冠脉流出液中乳酸脱氢酶(LDH)和肌酸激酶(CK)的变化。结果再灌注后,与对照组相比,ET-1组的LVDP明显升高,LVEDP显著降低,CF也稍有升高。ET-1组再灌注5 min后的LDH、CK较对照组明显下降,而且,低剂量ET-1组(0.1、1 nmol/L)效果更显著。但是,ET-1组的效果被BQ-123所抑制。结论 ET-1对离体心脏缺血再灌注损伤具有一定的保护作用,且低剂量时效果更显著,这种保护作用可能是由ETA受体所介导。     

卵巢癌细胞中RNF123对p27Kip1蛋白稳定性的调控作用

目的:研究卵巢癌细胞中RNF123对p27Kip1蛋白稳定性的调控作用?方法:流式细胞分析仪测定血清饥饿释放过程中SKOV3细胞的周期分布情况,利用Western blot检测该过程中干扰RNF123前后p27Kip1蛋白的表达水平?在SKOV3细胞中转染sictrl和siRNF123,Western blot检测p27Kip1的半衰期?结果:SKOV3细胞血清饥饿48 h,SKOV3细胞周期阻滞在G1/G0期,而血清释放后S期显著增加?在此过程中,p27Kip1表达下调?转染siRNF123组相较于sictrl组,p27Kip1蛋白水平增高?降低RNF123表达后,p27Kip1的半衰期延迟?结论:在卵巢癌细胞中降低RNF123的表达能抑制p27Kip1的降解?