Life-Long Effects of Perinatal Asphyxia on Stress-Induced Proteins and Dynamin 1 in Rat Brain

In previous work, we have shown that perinatal asphyxia (PA) in the rat leads to life-long neurotransmitter deficits and impairment of cognitive functions and behavior. This observation made us examine protein expression in hippocampus of rats with PA at the end of the life span. We applied a well-documented and characterized animal model of PA. Pups, normoxic and asphyxiated for 20 min, were brought up until the age of 24 months and then were sacrificed. Hippocampal tissue was dissected from the brains, and proteins were run on two-dimensional gel electrophoresis with in-gel digestion and subsequent identification of proteins by MALDI-TOF followed by quantification of protein spots by specific software. In hippocampus of rats with PA, the stress proteins protein disulfide isomerase A3 precursor and stress-induced phosphoprotein-1 were significantly increased, whereas the microtubule-associated protein dynamin-1 was significantly reduced. Increased stress protein levels may represent long-term effects of PA or, alternatively, could reflect conditioning of the stress protein machinery known to occur as a neuroprotective principle following hypoxic-ischemic conditions. Decreased dynamin-1 levels may be considered as a long-term effect on the exocytotic system possibly reflecting or leading to impaired neuronal transport and vesicle-trafficking in PA of the rat of advanced age.     

Chimeric influenza virus hemagglutinin proteins containing large domains of the Bacillus anthracis protective antigen: protein characterization, incorporation into infectious influenza viruses, and antigenicity

Large polypeptides of the Bacillus anthracis protective antigen (PA) were inserted into an influenza A virus hemagglutinin glycoprotein (HA), and the chimeric proteins were functionally characterized and incorporated into infectious influenza viruses. PA domain 1', the region responsible for binding to the other toxin components, the lethal factor and edema factor, and domain 4, the receptor binding domain (RBD), were inserted at the C-terminal flank of the HA signal peptide and incorporated into the HA1 subunit of HA. The chimeric proteins, designated as LEF/HA (90 amino acid insertion) and RBD/HA (140 amino acid insertion), were initially analyzed following expression using recombinant vaccinia viruses. Both chimeric proteins were shown to display functional phenotypes similar to that of the wild-type HA. They transport to the cell surface, can be cleaved into the HA1 and HA2 subunits by trypsin to activate membrane fusion potential, are able to undergo the low-pH-induced conformational changes required for fusion, and are capable of inducing the fusion process. We were also able to generate recombinant influenza viruses containing the chimeric RBD/HA and LEF/HA genes, and the inserted PA domains were maintained in the HA gene segments following several passages in MDCK cells or embryonated chicken eggs. Furthermore, DNA immunization of mice with plasmids that express the chimeric RBD/HA and LEF/HA proteins, and the recombinant viruses containing them, induced antibody responses against both the HA and PA components of the protein. These approaches may provide useful tools for vaccines against anthrax and other diseases.     

Complement C3d conjugation to anthrax protective antigen promotes a rapid, sustained, and protective antibody response

B. anthracis is the causative agent of anthrax. Pathogenesis is primarily mediated through the exotoxins lethal factor and edema factor, which bind protective antigen (PA) to gain entry into the host cell. The current anthrax vaccine (AVA, Biothrax) consists of aluminum-adsorbed cell-free filtrates of unencapsulated B. anthracis, wherein PA is thought to be the principle target of neutralization. In this study, we evaluated the efficacy of the natural adjuvant, C3d, versus alum in eliciting an anti-PA humoral response and found that C3d conjugation to PA and emulsion in incomplete Freund's adjuvant (IFA) imparted superior protection from anthrax challenge relative to PA in IFA or PA adsorbed to alum. Relative to alum-PA, immunization of mice with C3d-PA/IFA augmented both the onset and sustained production of PA-specific antibodies, including neutralizing antibodies to the receptor-binding portion (domain 4) of PA. C3d-PA/IFA was efficacious when administered either i.p. or s.c., and in adolescent mice lacking a fully mature B cell compartment. Induction of PA-specific antibodies by C3d-PA/IFA correlated with increased efficiency of germinal center formation and plasma cell generation. Importantly, C3d-PA immunization effectively protected mice from intranasal challenge with B. anthracis spores, and was approximately 10-fold more effective than alum-PA immunization or PA/IFA based on dose challenge. These data suggest that incorporation of C3d as an adjuvant may overcome shortcomings of the currently licensed aluminum-based vaccine, and may confer protection in the early days following acute anthrax exposure.     

Phosphatidylinositol Transfer Protein, Cytoplasmic 1 (PITPNC1) Binds and Transfers Phosphatidic Acid

Phosphatidylinositol transfer proteins (PITPs) are versatile proteins required for signal transduction and membrane traffic. The best characterized mammalian PITPs are the Class I PITPs, PITPα (PITPNA) and PITPβ (PITPNB), which are single domain proteins with a hydrophobic cavity that binds a phosphatidylinositol (PI) or phosphatidylcholine molecule. In this study, we report the lipid binding properties of an uncharacterized soluble PITP, phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) (alternative name, RdgBβ), of the Class II family. We show that the lipid binding properties of this protein are distinct to Class I PITPs because, besides PI, RdgBβ binds and transfers phosphatidic acid (PA) but hardly binds phosphatidylcholine. RdgBβ when purified from Escherichia coli is preloaded with PA and phosphatidylglycerol. When RdgBβ was incubated with permeabilized HL60 cells, phosphatidylglycerol was released, and PA and PI were now incorporated into RdgBβ. After an increase in PA levels following activation of endogenous phospholipase D or after addition of bacterial phospholipase D, binding of PA to RdgBβ was greater at the expense of PI binding. We propose that RdgBβ, when containing PA, regulates an effector protein or can facilitate lipid transfer between membrane compartments.     

1 alpha,25-dihydroxyvitamin D3 modulation in lipid metabolism in established bone marrow-derived stromal cells, MC3T3-G2/PA6.

MC3T3-G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] on adipogenesis in PA6 cells. When PA6 cells were cultured with 10(-8) M dexamethasone, adipocytes containing oil red O-positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1 alpha,25(OH)2D3 at 10(-9)M completely blocked this dexamethasone-induced neutral lipid synthesis throughout the 14-day culture period. Dose-response studies of vitamin D3 derivatives showed that 1 alpha,25(OH)2D3 was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]-acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10(-9) M 1 alpha,25(OH)2D3. Instead, 1 alpha,25(OH)2D3 greatly increased incorporation of [14C]-acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1 alpha,25(OH)2D3 modulation of lipid metabolism in bone marrow stromal cells is receptor mediated.     

Binding of phosphatidic acid to 14-3-3 proteins hampers their ability to activate the plant plasma membrane H+-ATPase

Phosphatidic acid is a phospholipid second messenger implicated in various cellular processes in eukaryotes. In plants, production of phosphatidic acid is triggered in response to a number of biotic and abiotic stresses. Here, we show that phosphatidic acid binds to 14-3-3 proteins, a family of regulatory proteins which bind client proteins in a phosphorylation-dependent manner. Binding of phosphatidic acid involves the same 14-3-3 region engaged in protein target binding. Consequently, micromolar phosphatidic acid concentrations significantly hamper the interaction of 14-3-3 proteins with the plasma membrane H(+)-ATPase, a well characterized plant 14-3-3 target, thus inhibiting the phosphohydrolitic enzyme activity. Moreover, the proton pump is inhibited when endogenous PA production is triggered by phospholipase D and the G protein agonist mastoparan-7. Hence, our data propose a possible mechanism involving PA that regulates 14-3-3-mediated cellular processes in response to stress.     

Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10?mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.     

Protein acylation in the cardiac muscle like cell line,H9c2

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with ³H-palmitate or ³H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 Ci palmitate (PA, 2.3 nmol) or 125 Ci myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 ± 0.25 to 1.25 ± 0.56 Ci/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 ± 0.12 to 0.77 ± 0.36 Ci/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with ³H-palmitate. Incorporation into phospholipids (PL) increased from 40–60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of ³H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.     

Phosphatidylinositol 3'-kinase-mediated calcium mobilization regulates chemotaxis in phosphatidic acid-stimulated human neutrophils

Phosphatidylinositol 3'-kinase (PI 3'-kinase) plays an important role in the migration of hepatocytes, endothelial cells and neoplastic cells to agonists which activate cellular tyrosine kinases. We examined the PI 3'-kinase-dependent chemotactic responses of neutrophilic leukocytes induced by phosphatidic acid (PA) in order to clarify mechanisms by which the enzyme potentially influences cellular migration. Western analysis of immunoprecipitates indicated that PA induced the tyrosine phosphorylation of three distinct proteins involved in functional activation which co-immunoprecipitated in PA-stimulated cells. These proteins were identified as lyn, syk and the 85 kDa regulatory subunit of PI 3'-kinase. Chemotactic responses to PA but not to several other neutrophil agonists were inhibited by the PI 3'-kinase inhibitors wortmannin and LY294002. Chemotactic inhibition resulted from upstream inhibition of calcium mobilization. Chelation of extracellular calcium by ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) did not affect the PA-induced chemotaxis, whereas chelation of intracellular calcium by 1, 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) attenuated this response. Thus, changes in intracellular Ca(2+) levels that can be effected by Ca(2+) mobilized from intracellular stores in the absence of Ca(2+) influx regulate PA-induced chemotaxis. Furthermore, PI 3'-kinase inhibition blunted the agonist-dependent generation of inositol 1,4,5-trisphosphate (IP(3)), suggesting that PI 3'-kinase exerted its effects on calcium mobilization from intracellular sources by mediating activation of phospholipase C (PLC) in PA-stimulated cells. Moreover, the PI 3'-kinase inhibitor LY294002 also inhibited phosphorylation of syk in PA-stimulated cells. We, therefore, propose that products of PI 3'-kinase confined to the inner leaflet of the plasma membrane play a role in activation of syk, calcium mobilization and induction of chemotactic migration.     

Proteome identification of binding-partners interacting with cell polarity protein Par3 in Jurkat cells

The evolutionarily conserved cell polarity protein Par3, a scaffold-like PDZreontaining protein, plays a critical role in the establishment and maintenance of epithelial cell polarity. Although the role of Par3 in establishing cell polarity in epithelial cells has been intensively explored, the function of Par3 in hematopoietic cells remains elusive. To address this issue, we generated GST-fusion proteins of Par3 PDZ domains. By combiningthe GST-pull-down approach with liquid chromatography-tandem mass spectrometry, we identified 10 potential novel binding proteins of PDZ domains of Par3 in Jurkat cells (a T-cell line). The interaction of Par3 with three proteins—nuclear transport protein importin-α4 and proteasome activators PA28β and PA28γ—was confirmed using in vitro binding assay, co-immunoprecipitation assay and immunofluorescence microscopy. Our results have the potential to uncover novel functions of the cell polarity protein Par3 in blood cells.     

Antiinflammatory and antiulcer activities of phytic acid in rats

Maximum antiinflammatory activity of phytic acid (PA) was seen at an oral dose of 150 mg/kg in the carrageenan induced rat paw edema model. Although PA showed ability to prevent denaturation of proteins, it showed less antiinflammatory activity than ibuprofen. Ability of PA to bring down thermal denaturation of proteins might be a contributing factor in the mechanism of action against inflammation. PA, at all the doses tested, showed significant protection from ulcers induced by ibuprofen, ethanol and cold stress, with a maximum activity at 150 mg/kg. There was a significant increase in gastric tissue malondialdehyde levels in ethanol treated rats but these levels decreased following PA pretreatment. Moreover, pretreatment with PA significantly inhibited various effects of ethanol on gastric mucosa, such as, reduction in the concentration of nonprotein sulfhydryl groups, necrosis, erosions, congestion and hemorrhage. These results suggested that gastro-protective effect of PA could be mediated by its antioxidant activity and cytoprotection of gastric mucosa.     

The 2beta2-2beta3 loop of anthrax protective antigen contains a dominant neutralizing epitope

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.     

The PI3K/AKT/mTOR Signaling Pathway Is Overactivated in Primary Aldosteronism

Background

To date, the available non-invasive remedies for primary aldosteronism are not satisfactory in clinical practice. The phosphoinositide 3-kinase (PI3Ks)/protein kinase B (PKB or AKT)/mammalian target of rapamycin (mTOR) signaling pathway is essential for tumorigenesis and metastasis in many types of human tumors, including renal cancer, adrenal carcinoma and pheochromocytoma. The possibility that this pathway is also necessary for the pathogenesis of primary aldosteronism has not yet been explored. To answer this question, we investigated the activity of the PI3K/AKT/mTOR signaling pathway in normal adrenal glands (NAGs), primary aldosteronism (PA) patients and NCI-H295R cells.

Methodology/Principal Findings

Between January 2005 and December 2011, we retrospectively reviewed the records of 45 patients with PA. We compared clinical characteristics (age, gender and biochemical data) and the expression of phospho-AKT (p-AKT), phospho-mTOR (p-mTOR), phospho-S6 (p-S6) and vascular endothelial growth factor (VEGF) by immunohistochemical staining and western blotting, analyzing 30 aldosterone-producing adenomas (APAs), 15 idiopathic hyperaldosteronism (IHA) tissues and 12 NAGs following nephrectomy for renal tumors (control group). Compared with the control group, most of the PA patients presented with polydipsia, polyuria, resistant hypertension, profound hypokalemia, hyperaldosteronemia and decreased plasma renin activity. Compared with normal zona glomerulosa, the levels of p-AKT, p-mTOR, p-S6 and VEGF were significantly upregulated in APA and IHA. No significant differences were found between APA and IHA in the expression of these proteins. Additionally, positive correlations existed between the plasma aldosterone levels and the expression of p-AKT and p-mTOR. In vitro studies showed that mTOR inhibitor rapamycin could inhibit cell proliferation in NCI-H295R cells in a dose- and time-dependent manner. Furthermore, this inhibitor also decreased aldosterone secretion.

Conclusions

Our data suggest that the PI3K/AKT/mTOR signaling pathway, which was overactivated in APA and IHA compared with normal zona glomerulosa, may mediate aldosterone hypersecretion and participate in the development of PA.     

High yield bacterial expression and purification of active recombinant PA28αβ complex

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon γ inducible PA28αβ, made of the two related subunits PA28α and β, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its β subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28α and β subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28αβ complexes, that remain soluble and can be easily purified by regular chromatographic procedures.     

Postmetamorphic changes in parvalbumin expression in the hindlimb skeletal muscle of the bullfrog,Rana catesbeiana

Anuran amphibians, animals that spend a terrestrial life after metamorphosis, exhibit a marked development of hindlimbs during and after metamorphosis. In order to see whether changes occur in the muscle protein components in the course of postmetamorphic development, we subjected gastrocnemius muscle extracts from growing froglets to two-dimensional electrophoresis (2DE). As a result, we found two proteins to undergo a change in level. One spot, indicating a molecular mass of approximately 12 kDa and an isoelectric point (pI) of 5.0 first became detectable at 45 days after metamorphosis. Another spot, corresponding to a protein of 11 kDa and pI 4.8, was prominent until the former spot appeared. N-terminal amino acid sequence analysis and comparison of the spots with those of parvalbumin (PA) revealed that these two proteins were PA alpha and PA beta. Northern blot analysis using PA alpha and PA beta cDNAs as probes revealed that the PA beta mRNA level declined whereas that of PA alpha mRNA rose as the frogs grew.     

Downregulation of sirtuin 3 by palmitic acid increases the oxidative stress,impairment of mitochondrial function,and apoptosis in liver cells

Elevated levels of saturated fatty acids show a strong cytotoxic effect in liver cells. Sirtuin 3 (SIRT3), a mitochondrially localized member of NAD⁺‐dependent deacetylase has been shown to protect hepatocytes against the oxidative stress. The role of SIRT3 on the cytotoxicity caused by fatty acids in liver cells is not fully understood. The aim of this study was to evaluate the expression level of SIRT3, oxidative stress, and mitochondrial impairments in human hepatoma HepG2 cells exposed to palmitic acid (PA). Our results showed that PA treatment caused the deposition of lipid droplets and resulted in an increased expression of tumor necrosis factor‐α in a dose‐dependent manner. Excessive accumulation of PA induces the reactive oxygen species formation and apoptosis while dissipating the mitochondrial transmembrane potential. The level of SIRT3 expression in both nuclear and mitochondrial fractions in HepG2 cells was decreased with the increase in PA concentrations. However, in the cytosolic fraction, the SIRT3 was undetectable. In conclusion, our results showed that PA caused an increase in inflammation and oxidative stress in HepG2 cells. The exposure of PA also resulted in the decline in transmembrane potential and an increase in apoptosis. The underexpression of nuclear and mitochondrial SIRT3 by PA suggests that the PA target the process that regulates the stress‐related gene expression and mitochondrial functions.