Cyclin-dependent kinase 5 prevents neuronal apoptosis by negative regulation of c-Jun N-terminal kinase 3

Cyclin-dependent kinase 5 (cdk5) is a serine/threonine kinase activated by associating with its neuron-specific activators p35 and p39. Analysis of cdk5(-/-) and p35(-/-) mice has demonstrated that both cdk5 and p35 are essential for neuronal migration, axon pathfinding and the laminar configuration of the cerebral cortex, suggesting that the cdk5-p35 complex may play a role in neuron survival. However, the targets of cdk5 that regulate neuron survival are unknown. Here, we show that cdk5 directly phosphorylates c-Jun N-terminal kinase 3 (JNK3) on Thr131 and inhibits its kinase activity, leading to reduced c-Jun phosphorylation. Expression of cdk5 and p35 in HEK293T cells inhibits c-Jun phosphorylation induced by UV irradiation. These effects can be restored by expression of a catalytically inactive mutant form of cdk5. Moreover, cdk5-deficient cultured cortical neurons exhibit increased sensitivity to apoptotic stimuli, as well as elevated JNK3 activity and c-Jun phosphorylation. Taken together, these findings show that cdk5 may exert its role as a key element by negatively regulating the c-Jun N-terminal kinase/stress-activated protein kinase signaling pathway during neuronal apoptosis.     

Cdk5 regulates the organization of Nestin and its association with p35

The intermediate filament protein nestin is characterized by its specific expression during the development of neuronal and myogenic tissues. We identify nestin as a novel in vivo target for cdk5 and p35 kinase, a critical signaling determinant in development. Two cdk5-specific phosphorylation sites on nestin, Thr-1495 and Thr-316, were established, the latter of which was used as a marker for cdk5-specific phosphorylation in vivo. Ectopic expression of cdk5 and p35 in central nervous system progenitor cells and in myogenic precursor cells induced elevated phosphorylation and reorganization of nestin. The kinetics of nestin expression corresponded to elevated expression and activation of cdk5 during differentiation of myoblast cell cultures and during regeneration of skeletal muscle. In the myoblasts, a disassembly-linked phosphorylation of Thr-316 indicated active phosphorylation of nestin by cdk5. Moreover, cdk5 occurred in physical association with nestin. Inhibition of cdk5 activity-either by transfection with dominant-negative cdk5 or by using a specific cdk5 inhibitor-blocked myoblast differentiation and phosphorylation of nestin at Thr-316, and this inhibition markedly disturbed the organization of nestin. Interestingly, the interaction between p35, the cdk5 activator, and nestin appeared to be regulated by cdk5. In differentiating myoblasts, p35 was not complexed with nestin phosphorylated at Thr-316, and inhibition of cdk5 activity during differentiation induced a marked association of p35 with nestin. These results demonstrate that there is a continuous turnover of cdk5 and p35 activity on a scaffold formed by nestin. This association is likely to affect the organization and operation of both cdk5 and nestin during development.     

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.     

Development of Novel Docetaxel Phospholipid Nanoparticles for Intravenous Administration: Quality by Design Approach

The objective of this study was to develop novel docetaxel phospholipid nanoparticles (NDPNs) for intravenous administration. Modified solvent diffusion-evaporation method was adopted in the NDPN preparation. Central composite design (CCD) was employed in the optimization of the critical formulation factor (drug content) and process variable (stirring rate) to obtain NDPNs with 215.53 ± 1.9-nm particle size, 0.329 ± 0.02 polydispersity index (PDI), and 75.41 ± 4.81% entrapment efficiency. The morphological examination by transmission electron microscopy revealed spherical structure composed of a drug core stabilized within the phospholipid shell. Enhanced cell uptake of coumarin-6-loaded phospholipid nanoparticles by MCF-7 cell line indicated NDPN-efficient cell uptake. In vitro hemolysis test confirmed the safety of the phospholipid nanoparticles. NDPNs exhibited increased area under the curve (AUC) and mean residence time (MRT) by 3.0- and 3.3-fold, respectively, in comparison with the existing docetaxel parenteral formulation (Taxotere®), indicating a potential for sustained action. Thus, the novel NDPNs exhibit an ability to be an intravenous docetaxel formulation with enhanced uptake, decreased toxicity, and prolonged activity.KEY WORDS: cancer, delivery vehicle, docetaxel phospholipid nanoparticles, nanoparticles, pharmacokinetics     

Human Social Behavior and Demography Drive Patterns of Fine-Scale Dengue Transmission in Endemic Areas of Colombia

Dengue is known to transmit between humans and A. aegypti mosquitoes living in neighboring houses. Although transmission is thought to be highly heterogeneous in both space and time, little is known about the patterns and drivers of transmission in groups of houses in endemic settings. We carried out surveys of PCR positivity in children residing in 2-block patches of highly endemic cities of Colombia. We found high levels of heterogeneity in PCR positivity, varying from less than 30% in 8 of the 10 patches to 56 and 96%, with the latter patch containing 22 children simultaneously PCR positive (PCR22) for DEN2. We then used an agent-based model to assess the likely eco-epidemiological context of this observation. Our model, simulating daily dengue dynamics over a 20 year period in a single two block patch, suggests that the observed heterogeneity most likely derived from variation in the density of susceptible people. Two aspects of human adaptive behavior were critical to determining this density: external social relationships favoring viral introduction (by susceptible residents or infectious visitors) and immigration of households from non-endemic areas. External social relationships generating frequent viral introduction constituted a particularly strong constraint on susceptible densities, thereby limiting the potential for explosive outbreaks and dampening the impact of heightened vectorial capacity. Dengue transmission can be highly explosive locally, even in neighborhoods with significant immunity in the human population. Variation among neighborhoods in the density of local social networks and rural-to-urban migration is likely to produce significant fine-scale heterogeneity in dengue dynamics, constraining or amplifying the impacts of changes in mosquito populations and cross immunity between serotypes.     

Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.