Algae permeability to Me(2)SO from -3 to 23 degrees C.

Biphasic transport of water and dimethyl sulfoxide (Me(2)SO), a common cryoprotective agent (CPA), in algal cells was induced and measured on a cryoperfusion stage. A two-step experimental protocol provided data for the volumetric response of Chlorococcum (C.) texanum to impermeable and permeable solutes. First, the cells were exposed to a 500-mOsm sucrose solution, causing immediate shrinkage of the cell to a minimum equilibrium volume. Then an isoosmotic 200-mOsm/300-mOsm CPA/sucrose solution was introduced to the cells, resulting in increased cell volume to a new equilibrium state. Experiments were conducted at temperatures between -3 and 23 degrees C. Cell volumes were measured off-line by computer analysis of video images. A network thermodynamic model was fit to the transient volume data to determine permeabilities of C. texanum to water and Me(2)SO over the full temperature range, and results were calculated with two numeric methods. Biphasic transport was found to be slower at colder temperatures, with water entering the cell faster than Me(2)SO. Experimental results were also compared with data from similar experiments using methanol (MeOH) as the CPA. MeOH influx was calculated to be a magnitude larger than that of water. Additionally, MeOH permeability was at least three orders of magnitude greater than Me(2)SO permeability, and the difference in these solute permeabilities increased as temperature decreased.     

Mitogenomic Analyses Place the Gharial (Gavialis gangeticus) on the Crocodile Tree and Provide Pre-K/T Divergence Times for Most Crocodilians

Based on morphological analyses, extant members of the order Crocodylia are divided into three families, Alligatoridae, Crocodylidae, and Gavialidae. Gavialidae includes one species, the gharial, Gavialis gangeticus. In this study we have examined crocodilian relationships in phylogenetic analyses of seven mitochondrial genomes that have been sequenced in their entirety. The analyses did not support the morphologically acknowledged separate position of the gharial in the crocodilian tree. Instead the gharial joined the false gharial (Tomistoma schlegelii) on a common branch that was shown to constitute a sister group to traditional Crocodylidae (less Tomistoma). Thus, the analyses suggest the recognition of only two Crocodylia families, Alligatoridae and Crocodylidae, with the latter encompassing traditional Crocodylidae plus Gavialis/Tomistoma. A molecular dating of the divergence between Alligatoridae and Crocodylidae suggests that this basal split among recent crocodilians took place ≈140 million years before present, at the Jurassic/Cretaceous boundary. The results suggest that at least five crocodilian lineages survived the mass extinction at the KT boundary. [Reviewing Editor: Dr. Nicolas Galtier]     

Correlation of HSP70 expression and cell viability following thermal stimulation of bovine aortic endothelial cells

Thermal preconditioning protocols for cardiac cells were identified which produce elevated HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44 to 50 degrees C for periods of 1-30 min. Thermal stimulation protocols were determined which induce HSP70 expression levels ranging from 2.3 to 3.6 times the control while maintaining cell viabilities greater than 90%. An Arrhenius injury model fit to the cell damage data yielded values of A = 1.4 X 10(66) s(-1) and Ea = 4.1 X 10(5) J/mol. Knowledge of the injury parameters and HSP70 kinetics will enhance dosimetry guideline development for thermal stimulation of heat shock proteins expression in cardiac tissue.     

Human DNA polymerase iota incorporates dCTP opposite template G via a G.C + Hoogsteen base pair

Human DNA polymerase iota (hPoliota), a member of the Y family of DNA polymerases, differs in remarkable ways from other DNA polymerases, incorporating correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. We present here the crystal structure of hPoliota bound to template G and incoming dCTP, which reveals a G.C + Hoogsteen base pair in a DNA polymerase active site. We show that the hPoliota active site has evolved to favor Hoogsteen base pairing, wherein the template sugar is fixed in a cavity that reduces the C1'-C1' distance across the nascent base pair from approximately 10.5 A in other DNA polymerases to 8.6 A in hPoliota. The rotation of G from anti to syn is then largely in response to this curtailed C1'-C1' distance. A G.C+ Hoogsteen base pair suggests a specific mechanism for hPoliota's ability to bypass N(2)-adducted guanines that obstruct replication.     

A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering beta1 integrin sialylation

Here we report that myeloid cells differentiating along the monocyte/macrophage lineage down-regulate the ST6Gal-I sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence, the beta1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of alpha5beta1 fibronectin receptors. Pharmacologic inhibitors of protein kinase C, Ras, and MEK, but not phosphoinositide 3-kinase, block ST6Gal-I down-regulation, integrin hyposialylation, and fibronectin binding. In contrast, constitutively active MEK stimulates these same events, indicating that ERK is both a necessary and sufficient activator of hyposialylation-dependent integrin activation. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified alpha5beta1 receptors bind fibronectin more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of these integrins with recombinant ST6Gal-I. Finally, we have mapped the N-glycosylation sites on the beta1 integrin to better understand the potential effects of differential sialylation on integrin structure/function. Notably, there are three N-glycosylated sites within the beta1 I-like domain, a region that plays a crucial role in ligand binding. Our collective results suggest that variant sialylation, induced by a specific signaling cascade, mediates the sustained increase in cell adhesiveness associated with monocytic differentiation.     

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.     

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.     

Honokiol potentiates apoptosis, suppresses osteoclastogenesis, and inhibits invasion through modulation of nuclear factor-kappaB activation pathway

Recent reports have indicated that honokiol can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this report, we found that honokiol potentiated the apoptosis induced by tumor necrosis factor (TNF) and chemotherapeutic agents, suppressed TNF-induced tumor cell invasion, and inhibited RANKL-induced osteoclastogenesis, all of which are known to require nuclear factor-kappaB (NF-kappaB) activation. Honokiol suppressed NF-kappaB activation induced by a variety of inflammatory stimuli, and this suppression was not cell type specific. Further studies showed that honokiol blocked TNF-induced phosphorylation, ubiquitination, and degradation of IkappaBalpha through the inhibition of activation of IkappaBalpha kinase and of Akt. This led to suppression of the phosphorylation and nuclear translocation of p65 and NF-kappaB-dependent reporter gene expression. Magnolol, a honokiol isomer, was equally active. The expression of NF-kappaB-regulated gene products involved in antiapoptosis (IAP1, IAP2, Bcl-x(L), Bcl-2, cFLIP, TRAF1, and survivin), proliferation (cyclin D1, cyclooxygenase-2, and c-myc), invasion (matrix metalloproteinase-9 and intercellular adhesion molecule-1), and angiogenesis (vascular endothelial growth factor) were also down-regulated by honokiol. Honokiol also down-regulated NF-kappaB activation in in vivo mouse dorsal skin model. Thus, overall, our results indicate that NF-kappaB and NF-kappaB-regulated gene expression inhibited by honokiol enhances apoptosis and suppresses osteoclastogenesis and invasion.     

Structural basis for error-free replication of oxidatively damaged DNA by yeast DNA polymerase η

7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase η (Polη). We show that the open active site cleft of Polη can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Polη to prevent the mutagenic replication of 8-oxoG lesions in cells.     

Neuroprotective effect of pioglitazone on acute phase changes induced by partial global cerebral ischemia in mice

Present study was carried out to investigate the possible neuroprotective effect of pioglitazone, an antidiabetic agent, peroxisome proliferator-activated receptor gamma (PPARgamma) agonist on acute phase changes in mice model of cerebral ischemia induced by Bilateral Common Carotid artery Occlusion (BCCAO). BCCAO model was used to induce partial global cerebral ischemia. BCCAO induced significant brain infarct size and edema in saline treated control group along with high increase in oxidative stress showed by increase lipid peroxidation and decreased levels of antioxidants like superoxide superoxide dismutage, catalase, glutathione peroxidase. Pioglitazone (20 mg/kg, orally) administration showed neuroprotective effects by reducing cerebral infarct size significantly as compared to control group. Postischemic seizure susceptibility was also reduced as number of positive responders decreased to a significant number. Brain edema was subsided to a significant level. Pioglitazone reduced the plasma TNF-alpha levels as compared to ischemia group significantly. Pioglitazone treatment also improved all the antioxidants levels showing activity against oxidative stress induced by BCCAO. Pioglitazone showed neuroprotection against ischemic insult suggesting the role of PPARgamma agonist in neuroprotective agents.