Nonspecific DNA binding and bending by HUαβ: interfaces of the three binding modes characterized by salt-dependent thermodynamics

Previous isothermal titration calorimetry (ITC) and Förster resonance energy transfer studies demonstrated that Escherichia coli HU_αβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34-bp mode that interacts with DNA in large (> 34 bp) gaps between bound proteins, reversibly bending it by 140^o and thereby increasing its flexibility, and two weaker, modestly cooperative small site-size modes (10 bp and 6 bp) that are useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and we deduce that DNA in the 34-bp mode is bent around—but not wrapped on—the body of HU, in contrast to specific binding of integration host factor. Analyses of binding isotherms (8-bp, 15-bp, and 34-bp DNA) and initial binding heats (34-bp, 38-bp, and 160-bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Sk_i) even though their binding site sizes differ greatly; the most probable values of Sk_i on 34-bp DNA or larger DNA are − 7.5 ± 0.5. From the similarity of Sk_i values, we conclude that the binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent DNA 34-bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6-bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Sk_i values are proposed.     

Expression and activation of an esterase from Pseudomonas aeruginosa 1001 in Escherichia coli

An expression vector was constructed to overproduce a maltose binding protein (MBP)-esterase fusion protein in Escherichia coli. Soluble fusion protein was separated by centrifugation after cell disruption. The fusion protein was partially purified with amylose resin. The higher concentration of fusion protein (above 2 mg/ml) did not show any activity but about 0.3 mg/ml of fusion protein had the highest activity (142 U/ml). It is due to the difficulty of contact between substrate and active site of enzyme in compact form at high concentration. The fusion protein over-expressed could not be separated into MBP and esterase by the action of protease ‘Factor Xa’. The esterase could be cleaved from MBP fusion protein by the treatment of SDS with the Factor Xa, and the resulting esterase activity was increased to 34% after cleavage.     

Induction of endothelial apoptosis by 4-hydroxyhexenal.

Lipid peroxidation and its products such as 4-hydroxy-2-nonenal (HNE) and 4-hydroxyhexenal (HHE) are known to affect redox balance during aging and various degenerative processes, including vascular dysfunction. Deterioration of the endothelial cells that line the vascular wall is known to be an underlying cause of vascular dysfunction. At present, little is known about the mechanism by which HHE induces endothelial cell death (i.e. apoptosis), although HNE-induced apoptotic cell death has been reported. The aim of this study was to determine whether apoptosis induced by HHE in endothelial cells involves peroxynitrite (ONOO(-)). Our results show that in endothelial cells HHE triggers apoptotic cell death by inducing apoptotic Bax coupled with a decrease in anti-apoptotic Bcl-2. Results show that HHE induces reactive oxygen species (ROS), nitric oxide, and ONOO(-) generation, leading to redox imbalance. Furthermore, the antioxidant N-acetyl cysteine, ROS scavenger, and penicillamine, an ONOO(-) scavenger, were found to block HHE-mediated apoptosis. We used confocal laser microscopy to estimate the ability of these inhibitors to attenuate HHE-induced intracellular ONOO(-) levels thus confirming the oxidative mediation of apoptosis in endothelial cells. These findings strongly suggest that accumulated HHE triggers reactive species-mediated endothelial apoptosis, leading to vascular dysfunction as well as vascular aging. During aging, increased lipid peroxidation and its associated production of HHE may exacerbate the weakened redox balance, leading to various chronic degenerative processes including vascular dysfunction.     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

Effect of apple intake on fecal microbiota and metabolites in humans

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.     

Intermittent Hypoxia Can Aggravate Motor Neuronal Loss and Cognitive Dysfunction in ALS Mice

Background

Patients with ALS may be exposed to variable degrees of chronic intermittent hypoxia. However, all previous experimental studies on the effects of hypoxia in ALS have only used a sustained hypoxia model and it is possible that chronic intermittent hypoxia exerts effects via a different molecular mechanism from that of sustained hypoxia. No study has yet shown that hypoxia (either chronic intermittent or sustained) can affect the loss of motor neurons or cognitive function in an in vivo model of ALS.

Objective

To evaluate the effects of chronic intermittent hypoxia on motor and cognitive function in ALS mice.

Methods

Sixteen ALS mice and 16 wild-type mice were divided into 2 groups and subjected to either chronic intermittent hypoxia or normoxia for 2 weeks. The effects of chronic intermittent hypoxia on ALS mice were evaluated using the rotarod, Y-maze, and wire-hanging tests. In addition, numbers of motor neurons in the ventral horn of the spinal cord were counted and western blot analyses were performed for markers of oxidative stress and inflammatory pathway activation.

Results

Compared to ALS mice kept in normoxic conditions, ALS mice that experienced chronic intermittent hypoxia had poorer motor learning on the rotarod test, poorer spatial memory on the Y-maze test, shorter wire hanging time, and fewer motor neurons in the ventral spinal cord. Compared to ALS-normoxic and wild-type mice, ALS mice that experienced chronic intermittent hypoxia had higher levels of oxidative stress and inflammation.

Conclusions

Chronic intermittent hypoxia can aggravate motor neuronal death, neuromuscular weakness, and probably cognitive dysfunction in ALS mice. The generation of oxidative stress with activation of inflammatory pathways may be associated with this mechanism. Our study will provide insight into the association of hypoxia with disease progression, and in turn, the rationale for an early non-invasive ventilation treatment in patients with ALS.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.     

Cigarette Smoking Increases Abdominal and Visceral Obesity but Not Overall Fatness: An Observational Study

Background

Cigarette smoking and obesity are leading public health concerns. Both increase the risk for cardiovascular disease, cancer, and metabolic abnormalities. This study was conducted to assess the association between cigarette smoking and different types of obesity.

Methodology/Principal Findings

Two hundred eighty-three visitors to university hospitals located in four main provinces of South Korea were participated. All participants were classified as either current/past or never smokers and were divided into quartiles according to the total pack-years. Body mass index, waist circumference, total body fat percentage, and area of visceral and abdominal subcutaneous fat were measured. These results of each groups were compared. Waist circumference, and visceral fat area showed a J- or U-shaped association with total smoking amount during a lifetime. After restricting the analyses to past/current smokers, we found significant dose-dependent associations of smoking pack-years with abdominal and visceral obesity. Overall obesity measured by body mass index and total body fat percentage did not show such associations. Although current smokers clearly showed significant associations, we could not demonstrate these in past smokers, possibly because of the limited sample size.

Conclusions/Significance

Although smokers did not show significant difference in mean body mass index than those who never smoked, they showed more metabolically adverse fat distributions with increasing smoking amounts. This finding suggests that smoking is not beneficial for weight control. Therefore, smoking cessation and avoidance of smoking commencement should be addressed as important public health issues in preventing obesity and related complications.