Adenosine uptake sites in dog heart and brain; interaction with calcium antagonists

[3H] Nitrobenzylthioinosine (NBI) binding is characterized in dog heart and brain. Evidence is presented suggesting that [3H]NBI is binding to the adenosine uptake site in both tissues. Physiologic studies in open-chested dogs clearly demonstrate that NBI acts as a coronary vasodilator, consistent with an action at the adenosine uptake site. The binding is reversible, saturable and of high affinity (KD = 0.78 +/- .06 nM for heart and 0.52 +/- .05 nM for brain). Both dipyridamole and hexobendine are high potency inhibitors of [3H]NBI binding in heart and brain while other antihypertensives and vasodilators such as propranolol and nitroglycerin have no effect. The inhibition of [3H]NBI binding observed with dipyridamole was competitive indicating that both agents are acting at the same site. The dihydropyridine calcium antagonists also inhibited binding with a lower potency than the adenosine uptake blockers. Non-dihydropyridine calcium antagonists were much less potent in this regard. The inhibition of [3H]NBI binding observed with the dihydropyridine calcium antagonists was non-competitive suggesting that the calcium channel and adenosine uptake site may be coupled to each other.     

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.     

Congenital heart disease and external ear anomalies with hearing loss: a report of two new cases and a review of the literature

Two cases of infants presenting primarily with congenital heart disease and external ear anomalies with hearing loss are reported. There is a clear, clinically important, association between these two birth defects; this association discussed along with a review of other syndromes with such features.     

The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity

Metabolic rate and the subsequent production of reactive oxygen species are thought to contribute to the rate of aging in a wide range of species. The target of rapamycin (TOR) is a well conserved serine/threonine kinase that regulates cell growth in response to nutrient status. Here we demonstrate that in mammalian cells the mammalian TOR (mTOR) pathway plays a significant role in determining both resting oxygen consumption and oxidative capacity. In particular, we demonstrate that the level of complex formation between mTOR and one of its known protein partners, raptor, correlated with overall mitochondrial activity. Disruption of this complex following treatment with the mTOR pharmacological inhibitor rapamycin lowered mitochondrial membrane potential, oxygen consumption, and ATP synthetic capacity. Subcellular fractionation revealed that mTOR as well as mTOR-raptor complexes can be purified in the mitochondrial fraction. Using two-dimensional difference gel electrophoresis, we further demonstrated that inhibiting mTOR with rapamycin resulted in a dramatic alteration in the mitochondrial phosphoproteome. RNA interference-mediated knockdown of TSC2, p70 S6 kinase (S6K1), raptor, or rictor demonstrates that mTOR regulates mitochondrial activity independently of its previously identified cellular targets. Finally we demonstrate that mTOR activity may play an important role in determining the relative balance between mitochondrial and non-mitochondrial sources of ATP generation. These results may provide insight into recent observations linking the TOR pathway to life span regulation of lower organisms.     

Role of penicillin-binding protein 1b in competitive stationary-phase survival of Escherichia coli

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.     

Regulation of Autophagy by the p300 Acetyltransferase

Autophagy is a regulated process of intracellular catabolism required for normal cellular maintenance, as well as serving as an adaptive response under various stress conditions, including starvation. The molecular regulation of autophagy in mammalian cells remains incompletely understood. Here we demonstrate a role for protein acetylation in the execution and regulation of autophagy. In particular, we demonstrate that the p300 acetyltransferase can regulate the acetylation of various known components of the autophagy machinery. Knockdown of p300 reduces acetylation of Atg5, Atg7, Atg8, and Atg12, although overexpressed p300 increases the acetylation of these same proteins. Furthermore, p300 and Atg7 colocalize within cells, and the two proteins physically interact. The interaction between p300 and Atg7 is dependent on nutrient availability. Finally, we demonstrate that knockdown of p300 can stimulate autophagy, whereas overexpression of p300 inhibits starvation-induced autophagy. These results demonstrate a role for protein acetylation and particularly p300 in the regulation of autophagy under conditions of limited nutrient availability.Macro-autophagy, herein referred to as autophagy, is an evolutionary conserved process first characterized in lower organisms (1). In yeast, over 20 separate genes (designated ATG1, ATG2, etc.) have been demonstrated to be essential to carry out the autophagy program. This process is thought to provide a mechanism for the efficient removal of both long lived proteins and damaged cellular organelles. This regulated degradation provides several essential functions for the cell. First, it allows for the removal of damaged and potentially harmful cellular contents. In addition, in breaking down various intracellular components, the autophagy process provides essential building blocks for the cell to use in the re-synthesis of necessary macromolecules. To accomplish this recycling effort, the coordinated actions of various Atg gene products are required. In particular, the Atg gene products together orchestrate the formation of a double membrane structure known as the autophagosome that engulfs the intended cellular cargo targeted for degradation. The autophagosome eventually fuses with the vacuole in yeast or the lysosome in mammals.In both yeast and mammalian cells, autophagy can be stimulated by the withdrawal of nutrients. Under these conditions, autophagic degradation of nonessential components may be essential to meet ongoing energetic needs in the presence of limited extracellular nutrients. This point was underscored by the analysis of mice containing a targeted deletion of Atg5 (2). In the absence of Atg5, there is a lack of both basal and starvation-induced autophagy. Mice lacking Atg5 are born normally but succumb within the 1st day of life. This post-natal lethality is thought to be due in large part for the requirement of autophagy to supply the energetic needs of neonates. These needs are particularly critical during the small window of time where the animal no longer has a placental circulation and before the pup can begin to nurse and thus obtain external nutrients.Relatively little is known regarding how signals such as nutrient availability are able to be transduced to ultimately regulate the level of cellular autophagy. One important pathway that impinges on the process is signaling thorough the target of rapamycin (TOR)2 network (3). Evidence suggests that TOR signaling inhibits autophagy, and indeed agents such as rapamycin that can inhibit TOR are known to result in increased autophagy. We recently have observed that in addition to this mode of regulation, the NAD-dependent deacetylase Sirt1 is also a regulator of autophagy in mammalian cells and tissues (4). In particular, we demonstrated that in the absence of Sirt1 levels of acetylation for various components of the autophagy machinery are increased and that starvation-induced autophagy is impaired. Interestingly, like the Atg5 knock-out animals, Sirt1^-/- mice are also born normally but die within the few hours to days after birth. Consistent with a defect in autophagy, electron micrographs of hearts from Sirt1^-/- mice demonstrated an accumulation of abnormal appearing organelles, including mitochondria, a phenotype previously observed in Atg-deficient animals (5). Here we have further characterized the role of acetylation in the regulation of autophagy, and in particular, we demonstrate a role for the p300 acetyltransferase in this process.     

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Background

Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.

Methods and Findings

A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient''s peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.

Conclusions

This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.     

Antiglycation Effects of Carnosine and Other Compounds on the Long-Term Survival of Escherichia coli

Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer''s disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.One factor that may affect the long-term survival of bacterial cells in a population is the level of damage incurred by macromolecules via the nonenzymatic process of glycation, first described by Louis-Camille Maillard (16). The Maillard reaction is responsible for the formation of several compounds identified as advanced glycation end products (AGEs) (9). In vivo this reaction appears to play a role in the aging process, as it leads to slow degradation of molecules. The principal mechanisms of glycation-related damage involve cross-links between proteins and/or DNA, modifying or destroying their functional properties (2, 8, 38). Most studies of glycation have been performed with eukaryotes because of its relationship to aging and disorders such as Alzheimer''s disease and diabetes (6, 21, 30, 42). However, several studies (32, 33) have shown that glycation also takes place in Escherichia coli, affecting protein and DNA of this prokaryote.Many biochemical pathways produce reactive dicarbonyl intermediates, such as glyoxal and methylglyoxal (MG), which can further react with DNA, proteins, or other biomolecules to form AGEs (8, 36). Reaction of glucose with amino groups of proteins and subsequent formation of reactive dicarbonyls via a series of reactions involving Schiff base and Amadori product intermediates have been well documented (40). Methylglyoxal can be formed by spontaneous decomposition of glycolytic triose phosphates such as dihydroxyacetone phosphate (DHAP) (1) or can be produced enzymatically from DHAP by the E. coli enzyme methylglyoxal synthase (MgsA) (12). MG synthesis usually requires an environment low in phosphate and high in DHAP, a situation that occurs most frequently under high-glucose conditions (25, 26). If MG is not degraded, MG accumulation will lead to cell death (12). E. coli maintains pathways for the detoxification of methylglyoxal, including glyoxalase enzymes I and II (encoded by gloA and gloB, respectively), which convert MG to S-lactoyl glutathione and then to d-lactate (12). This system has been proposed to be the predominant MG detoxification system in E. coli (12, 29).Glyoxal is also a toxic dicarbonyl compound capable of damaging cells via AGE formation. One of the AGEs formed in the presence of glyoxal is carboxymethyl lysine (CML), which has been used extensively as a biomarker for aging (11, 20, 31, 39). CML can be formed by different pathways: glucose can be oxidized to glyoxal, which can react with protein to form CML (1, 17); glucose can also react with protein to form fructoselysine (an Amadori product), which can undergo oxidative cleavage to form CML (1). In this study, we investigated CML formation in E. coli growing under standard and glycation-prone laboratory conditions. Since AGE formation may negatively affect cell survival and reproduction during long-term batch culture (35), we hypothesized that CML would accumulate in these cultures as cells progress through stationary phase.One product that may interfere with AGE formation is carnosine (β-alanyl-l-histidine), a naturally occurring dipeptide in many organisms. Although its mechanism of action has not been fully determined, there is evidence that both the free amino group derived from the β-alanine and the imidazole ring of histidine compete with amino groups of proteins in the presence of reactive dicarbonyl compounds (7, 24). In this study we designed assays to determine the effect of carnosine (and other compounds) on survival of cultures of E. coli under a variety of experimental conditions. Additionally, since strains lacking glyoxalase enzymes I and II have a reduced ability to detoxify methylglyoxal, we hypothesized that gloA and/or gloB mutants would require larger amounts of carnosine than would wild-type strains to survive in the presence of this toxic electrophile.