Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-β1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.     

Small amounts of isotope-reinforced polyunsaturated fatty acids suppress lipid autoxidation

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast.     

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS-internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent-resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome-like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS-exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.     

Solitary restriction endonucleases in prokaryotic genomes

Prokaryotic restriction-modification (R-M) systems defend the host cell from the invasion of a foreign DNA. They comprise two enzymatic activities: specific DNA cleavage activity and DNA methylation activity preventing cleavage. Typically, these activities are provided by two separate enzymes: a DNA methyltransferase (MTase) and a restriction endonuclease (RE). In the absence of a corresponding MTase, an RE of Type II R-M system is highly toxic for the cell. Genes of the R-M system are linked in the genome in the vast majority of annotated cases. There are only a few reported cases in which the genes of MTase and RE from one R-M system are not linked. Nevertheless, a few hundreds solitary RE genes are present in the Restriction Enzyme Database (http://rebase.neb.com) annotations. Using the comparative genomic approach, we analysed 272 solitary RE genes. For 57 solitary RE genes we predicted corresponding MTase genes located distantly in a genome. Of the 272 solitary RE genes, 99 are likely to be fragments of RE genes. Various explanations for the existence of the remaining 116 solitary RE genes are also discussed.     

Inhibition of renin activity slows down the progression of HIV-associated nephropathy

In the present study, we evaluated the effect of inhibition of renin activity (aliskiren) on the progression of renal lesions in two different mouse models (Vpr and Tg26) of human immunodeficiency virus (HIV)-associated nephropathy (HIVAN). In protocol A, Vpr mice were fed either water (C-VprA) or doxycycline [Doxy (D-VprA)] in their drinking water for 6 wk. In protocols B and C, Vpr mice received either normal saline (C-VprB/C), Doxy + normal saline (D-VprB/C), or Doxy + aliskiren (AD-VprB/C) for 6 wk (protocol B) or 12 wk (protocol C). In protocols D and E, Vpr mice were fed Doxy for 6 wk followed by kidney biopsy. Subsequently, half of the mice were administered either normal saline (D-VprD/E) or aliskiren (AD-VprD/E) for 4 wk (protocol D) or 8 (protocol E) wk. All D-VprA mice showed renal lesions in the form of focal segmental glomerular sclerosis and dilatation of tubules. In protocols B and C, aliskiren diminished both progression of renal lesions and proteinuria. In protocol C, aliskiren also diminished (P < 0.01) the rise in blood urea. In all groups, Doxy-treated mice displayed increased serum ANG I levels (the product of plasma renin activity); on the other hand, all aliskiren-treated mice displayed diminished serum ANG I levels. Renal tissues of D-VprC displayed increased ANG II content; however, aliskiren attenuated renal tissue ANG II production in AD-VprC. In protocol D, AD-VprD showed a 24.2% increase in the number of sclerosed glomeruli compared with 139.2% increase in sclerosed glomeruli in D-VprD (P < 0.01) from their baseline. The attenuating effect of aliskiren on the progression of renal lesions continued in AD-VprE. Aliskiren also diminished blood pressure, proteinuria, and progression of renal lesions in Tg26 mice. These findings indicate that inhibition of renin activity has a potential to slow down the progression of HIVAN.     

PET amyloid-beta imaging in preclinical Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of dementia, accounting for 60-70% of all cases [Hebert et al., 2003, 1]. The need for effective therapies for AD is great. Current approaches, including cholinesterase inhibitors and N-methyl-d-aspartate (NMDA) receptor antagonists, are symptomatic treatments for AD but do not prevent disease progression. Many diagnostic and therapeutic approaches to AD are currently changing due to the knowledge that underlying pathology starts 10 to 20 years before clinical signs of dementia appear [Holtzman et al., 2011, 2]. New therapies which focus on prevention or delay of the onset or cognitive symptoms are needed. Recent advances in the identification of AD biomarkers now make it possible to detect AD pathology in the preclinical stage of the disease, in cognitively normal (CN) individuals; this biomarker data should be used in the selection of high-risk populations for clinical trials. In vivo visualization of AD neuropathology and biological, biochemical or physiological confirmation of the effects of treatment likely will substantially improve development of novel pharmaceuticals. Positron emission tomography (PET) is the leading neuroimaging tool to detect and provide quantitative measures of AD amyloid pathology in vivo at the early stages and follow its course longitudinally. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.     

Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.     

Electric Field Exposure Triggers and Guides Formation of Pseudopod-Like Blebs in U937 Monocytes

We describe a new phenomenon of anodotropic pseudopod-like blebbing in U937 cells stimulated by nanosecond pulsed electric field (nsPEF). In contrast to “regular,” round-shaped blebs, which are often seen in response to cell damage, pseudopod-like blebs (PLBs) formed as longitudinal membrane protrusions toward anode. PLB length could exceed the cell diameter in 2 min of exposure to 60-ns, 10-kV/cm pulses delivered at 10–20 Hz. Both PLBs and round-shaped nsPEF-induced blebs could be efficiently inhibited by partial isosmotic replacement of bath NaCl for a larger solute (sucrose), thereby pointing to the colloid-osmotic water uptake as the principal driving force for bleb formation. In contrast to round-shaped blebs, PLBs retracted within several minutes after exposure. Cells treated with 1 nM of the actin polymerization blocker cytochalasin D were unable to form PLBs and instead produced stationary, spherical blebs with no elongation or retraction capacity. Live cell fluorescent actin tagging showed that during elongation actin promptly entered the PLB interior, forming bleb cortex and scaffold, which was not seen in stationary blebs. Overall, PLB formation was governed by both passive (physicochemical) effects of membrane permeabilization and active cytoskeleton assembly in the living cell. To a certain extent, PLB mimics the membrane extension in the process of cell migration and can be employed as a nonchemical model for studies of cytomechanics, membrane–cytoskeleton interaction and cell motility.