Inducible nitric oxide synthase expression and plasma bilirubin changes in rats under intermittent hypoxia treatment

It has been reported that intermittent hypoxia treatment prevents oxidative injuries to the brain and protects the heart against ischemia-reperfusion injury. Both anti-oxidative defensive systems and prevention of free intracellular calcium overload might be the result of intermittent hypoxia. Thus, the purpose of this study was to explore the effects of intermittent hypoxia (8 h at 12 % O2 per day) for 0, 7 or 14 days on inducible nitric oxide synthase (iNOS) expression in the spleen and on splenic calcium response to the mitogen phytohemagglutinin (PHA). The results demonstrated that administration of intermittent hypoxia for 7 days caused severe hemolysis of erythrocytes in the spleen and the hemolytic condition was ameliorated by intermittent hypoxia for 14 days. However, a significant decline in splenic weight and an increase in plasma total bilirubin levels appeared in rats after hypoxia for 14 days. No calcium response to PHA was observed in splenocytes obtained from rats after intermittent hypoxia for 7 days. After intermittent hypoxia for 14 days, the calcium response to PHA was restored to the level of the controls. Intermittent hypoxia for 7 days was able to induce higher iNOS expression in splenic tissues than hypoxia for 14 days. These results suggested that intermittent hypoxia for 14 days appeared to involve acclimatization that protects the rats from oxidative injury through less hemolysis and iNOS expression in splenic tissues and by the presence of more bilirubin in the plasma. The increase in plasma total bilirubin levels might be the cause of induced adaptation to chronic intermittent hypoxia.     

Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation

Summary Infarction in adult rat brain was induced by middle cerebral arterial occlusion (MCAO) followed by reperfusion to examine whether taxifolin could reduce cerebral ischemic reperfusion (CI/R) injury. Taxifolin administration (0.1 and 1.0 μg/kg, i.v.) 60 min after MCAO ameliorated infarction (by 42%±7% and 62%±6%, respectively), which was accompanied by a dramatic reduction in malondialdehyde and nitrotyrosine adduct formation, two markers for oxidative tissue damage. Overproduction of reactive oxygen species (ROS) and nitric oxide (NO) via oxidative enzymes (e.g., COX-2 and iNOS) was responsible for this oxidative damage. Taxifolin inhibited leukocyte infiltration, and COX-2 and iNOS expressions in CI/R-injured brain. Taxifolin also prevented Mac-1 and ICAM-1 expression, two key counter-receptors involved in firm adhesion/transmigration of leukocytes to the endothelium, which partially accounted for the limited leukocyte infiltration. ROS, generated by leukocytes and microglial cells, activated nuclear factor-kappa B (NF-κB) that in turn signaled up-regulation of inflammatory proteins. NF-κB activity in CI/R was enhanced 2.5-fold over that of sham group and was inhibited by taxifolin. Production of both ROS and NO by leukocytes and microglial cells was significantly antagonized by taxifolin. These data suggest that amelioration of CI/R injury by taxifolin may be attributed to its anti-oxidative effect, which in turn modulates NF-κB activation that mediates CI/R injury. Yea-Hwey Wang, Wen-Yen Wang, Chia-Che Chang, and Kuo-Tong Liou contributed equally to this work.     

Pin1 Catalyzes Conformational Changes of Thr-187 in p27Kip1 and Mediates Its Stability through a Polyubiquitination Process

The cis-trans peptidylprolyl isomerase Pin1 plays a critical role in regulating a subset of phosphoproteins by catalyzing conformational changes on the phosphorylated Ser/Thr-Pro motifs. The phosphorylation-directed ubiquitination is one of the major mechanisms to regulate the abundance of p27^Kip1. In this study, we demonstrate that Pin1 catalyzes the cis-trans conformational changes of p27^Kip1 and further mediates its stability through the polyubiquitination mechanism. Our results show that the phosphorylated Thr-187-Pro motif in p27^Kip1 is a key Pin1-binding site. In addition, NMR analyses show that this phosphorylated Thr-187-Pro site undergoes conformational change catalyzed by Pin1. Moreover, in Pin1 knock-out mouse embryonic fibroblasts, p27^Kip1 has a shorter lifetime and displays a higher degree of polyubiquitination than in Pin1 wild-type mouse embryonic fibroblasts, suggesting that Pin1 plays a critical role in regulating p27^Kip1 degradation. Additionally, Pin1 dramatically reduces the interaction between p27^Kip1 and Cks1, possibly via isomerizing the cis-trans conformation of p27^Kip1. Our study thus reveals a novel regulatory mechanism for p27^Kip1 stability and sheds new light on the biological function of Pin1 as a general regulator of protein stability.Cellular differentiation and cell cycle inhibition are tightly controlled via sensitive molecular mechanisms. p27^Kip1, a member of the Cip/Kip family, is an essential cell cycle inhibitor that functions largely during the G₀/G₁ phase where it promotes the assembly of the cyclin D1-CDK4 complex and inhibits the kinase activity of the cyclin E-CDK2 complex in the G₁-S phase (1–4). Several review articles have elegantly summarized and discussed the detailed cellular functions of p27^Kip1 (1–6). p27^Kip1 is also a phosphoprotein with multiple Ser/Thr phosphorylation sites, including Ser-10, Ser-178, and Thr-187, followed by a proline residue. Hence, these motifs are potential substrate sites for proline-directed kinases (5, 6). Compared with Ser-178, which has not yet been well studied, the phosphorylation of Ser-10 and Thr-187 has been well characterized to be important for the regulation of p27^Kip1 function. For instance, Ser-10 has been found to be the major phosphorylation site of p27^Kip1 (7) and to play an important role in regulating cell migration (8–10), although the regulation of Ser-10 phosphorylation is still not completely defined (11, 12).In contrast to Ser-10 and Thr-178, Thr-187 is the best characterized phosphorylation site on p27^Kip1 and is known to regulate the complex formation of p27^Kip1-cyclin E-CDK2 (1–2). In addition, it is also widely accepted that Thr-187 plays a crucial role in determining the abundance of mature p27^Kip1 proteins. The phosphorylation of Thr-187 directs p27^Kip1 to an SCF^Skp2 ubiquitin ligase complex (consisting of Skp2-Skp1-Cks1-Cul1-Roc1), which in turn promotes the polyubiquitination and degradation of p27^Kip1 (13, 14). The crystal structure of the Skp1-Skp2-Cks1-p27^Kip1 phosphopeptide complex shows that p27^Kip1 binds both Cks1 and Skp2 and that the C terminus of Skp2 and Cks1 forms the substrate recognition core of the SCF complex (15). Furthermore, the structure of this complex has revealed that the phosphorylation of Thr-187 in p27^Kip1 is recognized by the phosphate-binding site of Cks1, indicating that Cks1 is not only a facilitator but also an indispensable component in p27^Kip1 degradation machinery (15).Pin1 is a unique peptidyl-prolyl isomerase (PPIase)² that recognizes only the phosphorylated Ser/Thr motif preceding a proline residue (16). In addition, Pin1 is very prominent in isomerizing the cis-trans conformation of prolyl-peptidyl bonds in its substrates, resulting in either the modification of their function (e.g. c-Jun (17), β-catenin (18), Bax (19), and Notch1 (20)) or modulation of their stability (e.g. cyclin D1 (21), p53 (22, 23), and NF-κB (24)). Loss of Pin1 in mice results in several phenotypes similar to those of cyclin D1-null mice (21) and neuronal degenerative phenotypes (25–28), suggesting the conformational changes mediated by Pin1 may be crucial for the normal functioning of cells. Additionally, Pin1 also plays important roles in cancer and other cellular events, which have been extensively discussed in several recent review articles (29–33).In this study, we show that Pin1 binds to p27^Kip1, mainly through the phosphorylated Thr-187-Pro motif, and causes subsequent prolyl isomerization of this cell cycle protein. Moreover, we also find that Pin1 can protect p27^Kip1 from degradation. Importantly, we demonstrate that by catalyzing conformational changes in p27^Kip1, Pin1 hinders its association with Cks1, resulting in a reduction of polyubiquitination of p27^Kip1 and protecting its degradation by SCF^Skp2 complexes. Our results suggest that the cis-trans isomerization catalyzed by Pin1 represents a novel regulatory mechanism during post-phosphorylation of proteins and polyubiquitination-directed degradation pathways.     

Precise pattern of recombination in serotonergic and hypothalamic neurons in a Pdx1-cre transgenic mouse line

Background

Multicellular organisms are characterized by a remarkable diversity of morphologically distinct and functionally specialized cell types. Transgenic techniques for the manipulation of gene expression in specific cellular populations are highly useful for elucidating the development and function of these cellular populations. Given notable similarities in developmental gene expression between pancreatic β-cells and serotonergic neurons, we examined the pattern of Cre-mediated recombination in the nervous system of a widely used mouse line, Pdx1-cre (formal designation, Tg(Ipf1-cre)89.1Dam), in which the expression of Cre recombinase is driven by regulatory elements upstream of the pdx1 (pancreatic-duodenal homeobox 1) gene.

Methods

Single (hemizygous) transgenic mice of the pdx1-cre^Cre/0 genotype were bred to single (hemizygous) transgenic reporter mice (Z/EG and rosa26R lines). Recombination pattern was examined in offspring using whole-mount and sectioned histological preparations at e9.5, e10.5, e11.5, e16.5 and adult developmental stages.

Results

In addition to the previously reported pancreatic recombination, recombination in the developing nervous system and inner ear formation was observed. In the central nervous system, we observed a highly specific pattern of recombination in neuronal progenitors in the ventral brainstem and diencephalon. In the rostral brainstem (r1-r2), recombination occurred in newborn serotonergic neurons. In the caudal brainstem, recombination occurred in non-serotonergic cells. In the adult, this resulted in reporter expression in the vast majority of forebrain-projecting serotonergic neurons (located in the dorsal and median raphe nuclei) but in none of the spinal cord-projecting serotonergic neurons of the caudal raphe nuclei. In the adult caudal brainstem, reporter expression was widespread in the inferior olive nucleus. In the adult hypothalamus, recombination was observed in the arcuate nucleus and dorsomedial hypothalamus. Recombination was not observed in any other region of the central nervous system. Neuronal expression of endogenous pdx1 was not observed.

Conclusions

The Pdx1-cre mouse line, and the regulatory elements contained in the corresponding transgene, could be a valuable tool for targeted genetic manipulation of developing forebrain-projecting serotonergic neurons and several other unique neuronal sub-populations. These results suggest that investigators employing this mouse line for studies of pancreatic function should consider the possible contributions of central nervous system effects towards resulting phenotypes.

     

Downregulation of TRAF2 Mediates NIK-Induced Pancreatic Cancer Cell Proliferation and Tumorigenicity

Background

Increased levels of NF-κB are hallmarks of pancreatic ductal adenocarcinoma (PDAC) and both classical and alternative NF-κB activation pathways have been implicated.

Methodology/Principal Findings

Here we show that activation of the alternative pathway is a source for the high basal NF-κB activity in PDAC cell lines. Increased activity of the p52/RelB NF-κB complex is mediated through stabilization and activation of NF-κB-inducing kinase (NIK). We identify proteasomal downregulation of TNF receptor-associated factor 2 (TRAF2) as a mechanism by which levels of active NIK are increased in PDAC cell lines. Such upregulation of NIK expression and activity levels relays to increased proliferation and anchorage-independent growth, but not migration or survival of PDAC cells.

Conclusions/Significance

Rapid growth is one characteristic of pancreatic cancer. Our data indicates that the TRAF2/NIK/NF-κB2 pathway regulates PDAC cell tumorigenicity and could be a valuable target for therapy of this cancer.     

Role of a propeller loop in the quaternary structure and enzymatic activity of prolyl dipeptidases DPP-IV and DPP9

The dipeptidyl peptidase (DPP) family members, including DPP-IV, DPP8, DPP9 and others, cleave the peptide bond after the penultimate proline residue and are drug target rich. The dimerization of DPP-IV is required for its activity. A propeller loop located at the dimer interface is highly conserved within the family. Here we carried out site-directed mutagenesis on the loop of DPPIV and identified several residues important for dimer formation and enzymatic activity. Interestingly, the corresponding residues on DPP9 have a different impact whereby the mutations decrease activity without changing dimerization. Thus the propeller loop seems to play a varying role in different DPPs.     

Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm? for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm? and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.     

Factors affecting lipid accumulation by <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> in a two-stage cultivation process

Reserve lipids of microalgae are promising for biodiesel production. However, optimization of cultivation conditions for both biomass yield and lipid production of microalgae is a contradictory problem because required conditions for both targets are different. In this study, a two-stage cultivation strategy is proposed to enhance lipid production of the microalga Nannochloropsis oculata. Biomass growth and lipid production were carried out in two separate and non-interacting stages. In first-stage cultivation, microalgae were cultivated in optimal conditions for cell growth. Then, microalgae were harvested and transferred into a growth-limited environment, thus enhancing lipid production of microalgae. Here, optimization of the lipid production stage (second stage) with respect to different levels of inoculum concentration, salinity of culture broth, and intensity of irradiance was performed. The results show that irradiance exhibits a significant influence on lipid production. The highest lipid productivity of 0.324 g L⁻¹ day⁻¹ was obtained with an inoculum concentration of 2.3 g L⁻¹, a salinity of 35 g L⁻¹, and an irradiance of 500 μmol photons m⁻² s⁻¹. The final yield of lipid obtained from the two-stage process was 2.82-times higher than that from traditional single-stage batch cultivation systems.