Separation of temperature sensitive and temperature insensitive components of the postsynaptic potentials in the frog motoneurons

Intracellular measurements were made in the in situ spinal cord of the frog at temperatures below 5 degrees C. Responses to volleys in the sciatic nerve, in the descending fibres and in the motor axons were studied. About 30% of the motoneurons responded to sciatic volleys with 1-3 ms segmental latency, which was short enough to assume electrotonic mediation of these responses. Another group of motoneurons responded with 6-8 ms latency, i.e. with the expected delay at chemical synapses at low temperature. Latency distribution of the sciatic-evoked postsynaptic potentials was clearly bimodal in contrast with that found at higher temperatures. Postsynaptic discharges occurred with rather long latency and they were attributed to chemically-mediated excitation. Some of the postsynaptic potentials to descending volleys also occurred with quite short latency, indicating possible electrotonic transmission from supraspinal centres to motoneurons. Latency distribution of the action potentials evoked from the motor axons was bimodal, corresponding to the different, i.e. antidromic and recurrent facilitatory, mechanism of these spikes. Calculated Q10 ratios for the sciatic-evoked reflex discharges and the afferent fibre volleys were about 2.3 and 1.8, respectively. We concluded that cooling helps to separate postsynaptic potentials according to their electrotonic and chemical mediation and that electrotonic excitation does not seem to have a primary role in the generation of postsynaptic discharges initiated by dorsal root volleys in the frog.     

Vaccination with Venezuelan equine encephalitis replicons encoding cowpox virus structural proteins protects mice from intranasal cowpox virus challenge

An anti-poxvirus vaccine based on replicon particles of Venezuelan equine encephalitis virus (VRP) is being developed. The cowpox virus genes encoding structural proteins corresponding to vaccinia virus proteins A33, B5, and A27 were each expressed from VRP. High serum IgG titers against these proteins were generated in BALB/c mice vaccinated with each of these VRP. VRP induced both IgG1 and IgG2a with a strong predominance of IgG2a production. The response is long-lasting, as evidenced by the retention of high anti-B5 serum IgG titers through at least 50 weeks after priming immunization. Mice vaccinated with B5-, A33- or A27-VRP individually or together survived intranasal challenge with cowpox virus, with the multivalent vaccine formulation providing more effective protection from weight loss and clinical signs of illness than the monovalent vaccines. These results demonstrate that VRP may provide an effective alternative to vaccinia virus vaccines against poxvirus infection.     

基于遗传算法的带有软时间窗的卫生车辆调度优化研究

采用遗传算法对应急医学救援过程中车辆调度进行了研究,考虑到救援物资需求的时间要求,建立了带有软时间窗的遗传算法数学模型。在简单遗传算法的基础上,针对车辆调度的特点,建立了科学的染色体编码方法,并对其中复制、交叉和变异进行了改进,提高了求解的效率和精度。最后采用实例对改进的遗传算法进行了测试,结果表明遗传算法在求解车辆调度问题上具有良好的性能,特别适合大规模应急医学救援车辆调度问题,表现出优越的智能化求解特点,能够为以后的应急医学救援车辆调度系统的开发提供决策优化模型。     

Ventral root depolarization and spinal reflex augmentation by a TRH analog in rat spinal cord

The experiments were performed on spinal rats transected at the C 1 level. Intravenous administrations of TRH and its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide), which has less endocrine activity, produced a marked increase in the monosynaptic reflex (MSR) and depolarization of the ventral root, without affecting the dorsal root reflex (DRR) and the resting potential of the dorsal root. Excitation of the monosynaptic reflex was not antagonized by chlorpromazine, haloperidol, atropine or cyproheptadine, and was not influenced by pretreatment with reserpine. The depolarization of the ventral root persisted in the presence of baclofen or tetrodotoxin. These findings suggest that depolarization of the ventral root induced by TRH and DN-1417 is due to a direct action on motoneuronal membranes, and that an increase in the monosynaptic reflex is in part due to a depolarization of motoneurones. The effect of DN-1417 were longer lasting than those of TRH.     

XIAP and P-glycoprotein co-expression is related to imatinib resistance in chronic myeloid leukemia cells

P-glycoprotein (Pgp) and XIAP co-expression has been discussed in the process of the acquisition of multidrug resistance (MDR) in cancer. Here, we evaluated XIAP and Pgp expression in chronic myeloid leukemia (CML) samples, showing a positive correlation between them. Furthermore, we evaluated the effects of imatinib in XIAP and Pgp expression using CML cell lines K562 (Pgp⁻) and K562-Lucena (Pgp⁺). Imatinib increased XIAP and Pgp expression in K562-Lucena cells, while in K562 cells a downregulation of these proteins was observed, suggesting that imatinib induces an increment of MDR phenotype of CML cells that previously exhibit high levels of Pgp/XIAP co-expression.     

Extracellular potassium accumulation and transmission in frog spinal cord

In the isolated frog spinal cord, intracellular recordings from motoneurons and neuroglia and extracellular recordings from dorsal and ventral roots were used to compare electrophysiological changes produced by tetanic stimulation of dorsal roots with those resulting from increasing extracellular potassium concentration, [K⁺]₀. Many of the after-effects of tetanic dorsal root stimulation could be mimicked by increasing [K⁺]₀. These include the following: depolarization of motoneurons and neuroglia, prolongation and depression of excitatory postsynaptic potentials, depression of dorsal root potentials, facilitation and depression of ventral root potentials, as well as increases in spontaneous synaptic activity and depression of antidromic spikes recorded from motoneurons. The levels of [K⁺]₀ necessary to produce effects comparable to those following maximal dorsal root stimulation are about twice those estimated from measurements with K-selective microelectrodes or glial depolarization, suggesting that both these methods underestimate the amount of potassium which accumulates in the narrow intercellular spaces between neurons and glia in the intermediate region of the spinal cord.It is concluded that, at least within the isolated frog spinal cord, K⁺ accumulation is a significant factor modifying transmission following dorsal root tetanic stimulation and that its distribution is inhomogeneous.     

Vectored co-delivery of human cytomegalovirus gH and gL proteins elicits potent complement-independent neutralizing antibodies

Human cytomegalovirus (hCMV) is prevalent worldwide with infection generally being asymptomatic. Nevertheless, hCMV infection can lead to significant morbidity and mortality. Primary infection of seronegative women or reactivation/re-infection of seropositive women during pregnancy can result in transmission to the fetus, leading to severe neurological defects. In addition, hCMV is the most common viral infection in immunosuppressed organ transplant recipients and can produce serious complications. Hence, a safe and effective vaccine to prevent hCMV infection is an unmet medical need.     

Modulation of GSH levels in ABCC1 expressing tumor cells triggers apoptosis through oxidative stress

The over-expression of ABCC1 transmembrane protein has been shown to cause multidrug resistance in tumor cell lines. ABCC1 is a member of the ABC transmembrane proteins that function as efflux pumps with diverse substrate specificity. Several endogenous cell metabolites, including the leukotriene C4 (LTC(4)) and glutathione (GSH) are substrates for ABCC1 protein. ABCC1 expression in certain tumor cells was demonstrated to confer hypersensitivity to glutathione modulating agents. In this report we have investigated the mechanism of collateral sensitivity seen in tumor cells over-expressing ABCC1 protein. The results of this study show that ABCC1 expression in tumor cells correlates with their hypersensitivity to various glutathione modulating agents, as demonstrated in H69AR-drug selected and HeLa/ABCC1-transfectant cells. This effect was triggered either through inhibition of GSH synthesis with BSO or by increasing ABCC1-mediated GSH transport with verapamil or apigenin. In addition, our results show that the hypersensitivity of ABCC1-expressing cells to BSO, verapamil or apigenin was preceded by an increase in reactive oxygen species (or ROS). A decrease in GSH level is also observed prior the increase in ROS. In addition, we show that hypersensitivity to the BSO, verapamil or apigenin leads to tumor cell death by apoptosis. Together, the results of this study demonstrate that ABCC1 potentiates oxidative stress in tumor cells through reductions in cellular GSH levels.     

Sensitization of ABCB1 overexpressing cells to chemotherapeutic agents by FG020326 via binding to ABCB1 and inhibiting its function

The effectiveness of chemotherapeutic treatment is usually limited by the overexpression of adenosine triphosphate binding cassette (ABC) transporters, which mediate multidrug resistance (MDR) by acting as efflux pumps to remove chemotherapeutic agents from MDR cancer cells. Thus, the inhibition of ABC transporters may represent a promising strategy to reverse MDR. This study was to characterize the actions of FG020326, a newly synthesized triaryl-substituted imidazole derivative, to reverse MDR in vitro and in vivo. FG020326 significantly potentiated the cytotoxicity of paclitaxel, doxorubicin, and vincristine in the ABCB1 (P-glycoprotein, P-gp) overexpressing cells KBv200 and MCF-7/adr, but not in the ABCB1 negative parental cell lines KB and MCF-7. However, FG020326 did not alter the cytotoxicity of the aforementioned drugs in ABCC1 (MRP1), ABCC4 (MRP4), ABCG2 (BCRP) and LRP overexpressing cell lines, KB-CV60, NIH3T3/MRP4-2, S1-M1-80 and SW1573/2R120, respectively. FG020326, following p.o. administration, was present in concentrations sufficient for reversal of MDR in mice. The co-administration of FG020326 with paclitaxel or vincristine significantly enhanced the antitumor activity of these drugs without significantly increasing toxicity in the mice bearing the KBv200 cell xenografts. In addition, FG020326, at concentrations that reversed MDR, did not significantly affect the activity of CYP3A4 or alter the pharmacokinetic profile of paclitaxel after co-administration with paclitaxel. FG020326 produced a significant concentration-dependent displacement of [³H]azidopine and inhibition of efflux of drug from cells. Furthermore, FG020326 was co-localized with ABCB1 in cell membranes. Hence, FG020326 is characterized as a third generation MDR modulator that holds great promise for the treatment of cancer patients with ABCB1-mediated MDR.     

VRP immunotherapy targeting neu: treatment efficacy and evidence for immunoediting in a stringent rat mammary tumor model

The ability to overcome intrinsic tolerance to a strict “self” tumor-associated antigen (TAA) and successfully treat pre-existing tumor is the most stringent test for anti-tumor immunotherapeutic strategies. Although this capacity has been demonstrated in various models using complicated strategies that may not be readily translated into the clinical arena, straightforward antigen-specific immunotherapeutic strategies in the most stringent models of common epithelial cancers have largely failed to meet this standard. We employed an immunotherapeutic strategy using an alphavirus-based, virus-like replicon particle (VRP), which has in vivo tropism for dendritic cells, to elicit immune responses to the non-mutated TAA rat neu in an aggressive rat mammary tumor model. Using this VRP-based immunotherapeutic strategy targeting a single TAA, we generated effective anti-tumor immunity in the setting of pre-existing tumor resulting in the cure of 36% of rats over multiple experiments, P = 0.002. We also observed down-regulation of rat neu expression in tumors that showed initial responses followed by tumor escape with resumption of rapid tumor growth. These responses were accompanied by significant anti-tumor proliferative responses and CD8+ cellular tumor infiltrates, all of which were restricted to animals receiving the anti-neu immunotherapy. Together these data, obtained in a stringent “self” TAA model, indicate that the VRP-based antigen-specific immunotherapy elicits sufficiently potent immune responses to exert immunologic pressure, selection, and editing of the growing tumors, thus supporting the activity of this straightforward immunotherapy and suggesting that it is a promising platform upon which to build even more potent strategies.