Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

p38-MAPK signaling pathway is not involved in osteogenic differentiation during early response of mesenchymal stem cells to continuous mechanical strain

Quercetin has been reported to protect testicular cells from oxidative damage induced by environmental chemicals. In this study, we isolated interstitial Leydig cells (ILCs) from immature rats, set-up ILCs culture, co-treated cells with atrazine (ATZ) and quercetin (QT), evaluated toxicity, and measured the expression levels of antioxidant enzymes and nuclear factor-kappaB (NF-κB) and levels of steroidogenic enzymes. ATZ decreased ILCs viability at concentrations higher than 10 μg/mL and increased reactive oxygen species, malondialdehyde (MDA), and glutathione levels. ATZ also increased glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and decreased superoxide dismutase-1 (sod1) and superoxide dismutase-2 (sod2) messenger RNA (mRNA) levels which were prevented by QT. The changes in the MDA levels and lactate dehydrogenase leakage induced by ATZ (50 μg/mL) were also prevented on co-treatment with QT (50 μM). Furthermore, ATZ-induced 3β- and 17β-hydroxysteroid dehydrogenase activities and NF-κB-expressions at the mRNA and protein levels were also recovered to control value on co-treatment with QT. These data showed that QT protected against ATZ-induced ILCs toxicity by restoring the expression of NF-κB and steroidogenic activity and by preventing the oxidative stress.     

π–π Stacking mediated drug–drug interactions in human CYP2E1

Because of having many low molecular mass substrates, CYP2E1 is of particular interests to the pharmaceutical industry. Many evidences showed that this enzyme can adopt multiple substrates to significantly reduce the oxidation rate of the substrates. The detailed mechanism for this observation is still unclear. In the current study, we employed GPU‐accelerated molecular dynamics simulations to study the multiple‐binding mode of human CYP2E1, with an aim of offering a mechanistic explanation for the unexplained multiple‐substrate binding. Our results showed that Thr303 and Phe478 were key factors for the substrate recognition and multiple‐substrate binding. The former can form a significant hydrogen bond to recognize and position the substrate in the productive binding orientation in the active site. The latter acted as a mediator for the substrate communications via π–π stacking interactions. In the multiple‐binding mode, the aforementioned π–π stacking interactions formed by the aromatic rings of both substrates and Phe478 drove the first substrate far away from the catalytic center, orienting in an additional binding position and going against the substrate metabolism. All these findings could give atomic insights into the detailed mechanism for the multiple‐substrate binding in human CYP2E1, providing useful information for the drug metabolism mechanism and personalized use of clinical drugs. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

ATM pathway is essential for ionizing radiation-induced autophagy

BackgroundATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells.MethodsHuman cervical cancer cells, Hela, were used, and cell models with ATM^?/? and MAPK14^?/? were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry.ResultsAfter radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM^?/? cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM^?/? and MAPK14^?/? cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclin's bond to PI3KIII and their interaction increased after IR, while in ATM^?/? and MAPK14^?/? cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM^?/? and MAPK14^?/? cells showed no interaction.ConclusionsATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.     

MAIGO2 is involved in abscisic acid‐mediated response to abiotic stresses and Golgi‐to‐ER retrograde transport

The central role of multisubunit tethering complexes in intracellular trafficking has been established in yeast and mammalian systems. However, little is known about their roles in the stress responses and the early secretory pathway in Arabidopsis. In this study, Maigo2 (MAG2), which is equivalent to the yeast Tip20p and mammalian Rad50‐interacting protein, is found to be required for the responses to salt stress, osmotic stress and abscisic acid in seed germination and vegetative growth, and MAG2‐like (MAG2L) is partially redundant with MAG2 in response to environmental stresses. MAG2 strongly interacts with the central region of ZW10, and both proteins are important as plant endoplasmic reticulum (ER)‐stress regulators. ER morphology and vacuolar protein trafficking are unaffected in the mag2, mag2l and zw10 mutants, and the secretory marker to the apoplast is correctly transported in mag2 plants, which indicate that MAG2 functions as a complex with ZW10, and is potentially involved in Golgi‐to‐ER retrograde trafficking. Therefore, a new role for ER–Golgi membrane trafficking in abiotic‐stress and ER‐stress responses is discovered.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

Ontogenic study of the brachiopod Dicoelosia by geometric morphometrics and morphing techniques

The distinctive brachiopod Dicoelosia King 1850 is characterized by a strongly bilobed outline. To date, studies have concentrated on its functional morphology, taxonomy and evolution; little attention has been paid to its ontogeny. Here, we map population variation by principal component analysis for over 80 specimens distributed across five species of Dicoelosia. Using geometric morphometrics with landmarks for some 40 specimens, the ontogenic trends in D. sp. nov. are compared with those of Dicoelosia biloba. In addition, the ontogenic pathway in D. sp. nov. is investigated by morphing with control points, a new technique introduced here to palaeontology. Combining the results above, the ontogeny of the key character of the genus, emargination, is modelled. Within single populations, taxa may develop from broad weakly emarginate forms into those that are elongate and deeply emarginate. As the identification of the genus and its species depends on external morphological characters, the definition of ontogenetic trends in each species is essential for taxonomic discrimination. Substantial population variation exists in many of its species; however, the morphing technique provides a method of simulation, predicting the full range of ontogenetic variation in given populations.     

AtSec20 is involved in osmotic stress tolerance and AtSec20 mutation unaffects the integrity of intracellular organelles and the anterograde biosynthetic trafficking

The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play essential roles in intracellular trafficking. However, few experimental data have clarified their roles in the stress responses and the early secretary pathway in Arabidopsis. The AtSec20 gene encodes a protein that is homologous to yeast Sec20p and mammalian BNIP1, which are involved in the Golgi-to-ER retrograde trafficking in yeast and mammalian cells. In this study, AtSec20 is found to be required for the responses to salt stress, osmotic stress and gibberellin (GA) during seed germination and early seedling establishment. Mutation of AtSec20 unaffects the morphology of intracellular organelles, such as endoplasmic reticulum (ER), trans-Golgi network, and peroxisome, and vacuolar protein trafficking is normal in sec20 mutants. Collectively, these results imply that the AtSec20 is involved in abiotic stress tolerance, potentially via roles in retrograde vesicle fusion process in Arabidopsis.