Differential, age-dependent MEK-ERK and PI3K-Akt activation by insulin acting as a survival factor during embryonic retinal development

Programmed cell death is a genuine developmental process of the nervous system, affecting not only projecting neurons but also proliferative neuroepithelial cells and young neuroblasts. The embryonic chick retina has been employed to correlate in vivo and in vitro studies on cell death regulation. We characterize here the role of two major signaling pathways, PI3K-Akt and MEK-ERK, in controlled retinal organotypic cultures from embryonic day 5 (E5) and E9, when cell death preferentially affects proliferating neuroepithelial cells and ganglion cell neurons, respectively. The relative density of programmed cell death in vivo was much higher in the proliferative and early neurogenic stages of retinal development (E3-E5) than during neuronal maturation and synaptogenesis (E8-E19). In organotypic cultures from E5 and E9 retinas, insulin, as the only growth factor added, was able to completely prevent cell death induced by growth factor deprivation. Insulin activated both the PI3K-Akt and the MEK-ERK pathways. Insulin survival effect, however, was differentially blocked at the two stages. At E5, the effect was blocked by MEK inhibitors, whereas at E9 it was blocked by PI3K inhibitors. The cells which were found to be dependent on insulin activation of the MEK-ERK pathway at E5 were mostly proliferative neuroepithelial cells. These observations support a remarkable specificity in the regulation of early neural cell death.     

Prevalence of endoparasitic mites on four West African leaf‐litter frogs depends on habitat humidity

Amphibian species are known to carry endoparasitic mites. The infestation probability, prevalence, and intensity of mites vary among species and habitats. Mites of the genus Endotrombicula are known to infest African and Malagasy frogs. However, the factors leading to an increase in the probability of mite infestation are unknown. To test for inter‐ and intraspecific differences in infestation probability and its potential correlation with sex, age, habitat preferences, and/or season within a species‐rich West African leaf‐litter frog assemblage, we examined more than 6,800 individual frogs for the presence of mites throughout two independent time increments, 1999–2000 and 2016–2017. We found only members of the leaf‐litter frog genus Phyrnobatrachus to be infested, while other syntopically occurring genera were not affected. Within Phrynobatrachus, only four out of eight species were infested. Mites prevalence differed between species (highest P. phyllophilus, followed by P. alleni), sex (males higher than females in P. alleni and P. phyllophilus), and age (adults higher than juveniles in P. alleni), as well as season (more mites during wet than dry season in P. phyllophilus). The prevalence of mite infestation did not influence mate choice in P. alleni. Increased humidity showed a clear positive effect on infestation prevalence. We also detected a marked decrease in the prevalence of mites from 1999–2000 to 2016–2017, a period during which climatological changes within the study area have been reported with a tendency toward drier conditions. The decrease in mite infestation prevalence over time might be a signal of increasingly drier conditions.     

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.     

Age-related increases of 8-hydroxy-2'-deoxyguanosine and DNA-protein crosslinks in mouse organs

Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.     

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Heat shock proteins (HSPs) are highly conserved among all organisms from prokaryotes to eukaryotes. In mice, the HSP genes Hsp70.1 and Hsp70.3 are induced by both endogenous and exogenous stressors, such as heat and toxicants. In order to determine whether such proteins specifically influence genomic instability, mice deficient for Hsp70.1 and Hsp70.3 (Hsp70.1/3(-/-) mice) were generated by gene targeting. Mouse embryonic fibroblasts (MEFs) prepared from Hsp70.1/3(-/-) mice did not synthesize Hsp70.1 or Hsp70.3 after heat-induced stress. While the Hsp70.1/3(-/-) mutant mice were fertile, their cells displayed genomic instability that was enhanced by heat treatment. Cells from Hsp70.1/3(-/-) mice also display a higher frequency of chromosome end-to-end associations than do control Hsp70.1/3(+/+) cells. To determine whether observed genomic instability was related to defective chromosome repair, Hsp70.1/3(-/-) and Hsp70.1/3(+/+) fibroblasts were treated with ionizing radiation (IR) alone or heat and IR. Exposure to IR led to more residual chromosome aberrations, radioresistant DNA synthesis (a hallmark of genomic instability), increased cell killing, and enhanced IR-induced oncogenic transformation in Hsp70.1/3(-/-) cells. Heat treatment prior to IR exposure enhanced cell killing, S-phase-specific chromosome damage, and the frequency of transformants in Hsp70.1/3(-/-) cells in comparison to Hsp70.1/3(+/+) cells. Both in vivo and in vitro studies demonstrate for the first time that Hsp70.1 and Hsp70.3 have an essential role in maintaining genomic stability under stress conditions.     

Lead-induced oxidative stress and hematological alterations and their response to combined administration of calcium disodium EDTA with a thiol chelator in rats

The therapeutic efficacy of calcium disodium ethylenediaminetetracetic acid (CaNa(2)EDTA) and the two thiol chelators, 2,3-dimercaptopropane 1-sulfonate (DMPS) and monoisoamyl dimercaptosuccinic acid (MiADMSA) was studied, both individually and in combination, in reducing lead concentration in blood and soft tissues and in restoring lead induced altered biochemical variables in rats. Exposure to subacute dose of lead implicated a critical role of reactive oxygen species (ROS) and oxidative stress in altering the normal values of these variables. Exposure to lead caused a significant inhibition of blood delta-aminolevulinic acid dehydratase (ALAD), an important enzyme in the haem synthesis pathway and glutathione (GSH) level. These changes were also accompanied by inhibition of ALAD activity in kidney, delta-aminolevulinic acid synthase (ALAS) activities in liver and changes in platelet counts in whole blood suggesting disturbed haem synthesis pathway. Lead exposure also led to a pronounced depletion of brain GSH contents, superoxide dismutase (SOD) activity, an increase in thiobarbituric acid reactive substances (TBARS), and activity of glutathione S-transferase (GST). Specific activities of membrane-bound enzymes, acetylcholinesterase (AChE) and monoamine oxidase (MAO), were significantly inhibited on lead exposure. These biochemical changes were correlated with increased uptake of lead in blood and soft tissues. Post lead exposure treatment with MiADMSA in particular provided significant recovery in altered biochemical variables besides significant depletion of tissue lead burden. Treatment with CaNa(2)EDTA and DMPS individually had only moderate beneficial effects on tissue oxidative stress, although they were equally effective in the removal of tissue lead burden. Tissue zinc and copper levels did not depict any significant depletion, although changes like marked depletion of zinc following CaNa(2)EDTA and copper after MiADMSA administration were of some concern. Combined administration of CaNa(2)EDTA, particularly with MiADMSA, was the most effective treatment protocol compared to all other treatments. It can be concluded from our present results that combined therapy with CaNa(2)EDTA and MiADMSA proved significantly better in restoring biochemical and clinical variables over monotherapy with these chelating agents against subacute lead exposure in adult rats.     

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(-/-)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(-/-) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(-/-) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.     

A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation

In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.     

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites

In partially denervated rodent muscle, terminal Schwann cells (TSCs) located at denervated end plates grow processes, some of which contact neighboring innervated end plates. Those processes that contact neighboring synapses (termed "bridges") appear to initiate nerve terminal sprouting and to guide the growth of the sprouts so that they reach and reinnervate denervated end plates. Studies conducted prior to knowledge of this potential involvement of Schwann cells showed that direct muscle stimulation inhibits terminal sprouting following partial denervation (Brown and Holland, 1979). We have investigated the possibility this inhibition results from an alteration in the growth of TSC processes. We find that stimulation of partially denervated rat soleus muscle does not alter the length or number of TSC processes but does reduce the number of TSC bridges. Stimulation also reduces the number of TSC bridges that form between end plates during reinnervation of a completely denervated muscle. The nerve processes ("escaped fibers") that normally grow onto TSC processes during reinnervation are also reduced in length. Therefore, stimulation alters at least two responses to denervation in muscles: (1) the ability of TSC processes to form or maintain bridges with innervated synaptic sites, and (2) the growth of axons along processes extended by TSCs.