Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors

The cellular pathways of apoptosis have not been fully characterized; however, calpain, a cytosolic calcium-activated cysteine protease, has been implicated in several forms of programmed cell death. Reoviruses induce apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of calpain in reovirus-induced apoptosis in vitro by measuring calpain activity as well as evaluating the effects of calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and calpain activity, measured as cleavage of the fluorogenic calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of apoptosis by calpain inhibitors is independent of effects on viral replication. We conclude that calpain activation is a component of the regulatory cascade in reovirus-induced apoptosis.     

Cohesin's DNA Exit Gate Is Distinct from Its Entrance Gate and Is Regulated by Acetylation

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal?a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.     

Distance between AER and ZPA Is Defined by Feed-Forward Loop and Is Stabilized by their Feedback Loop in Vertebrate Limb Bud

In the development of organs, multiple morphogen sources are often involved, and interact with each other. For example, the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) are major morphogen sources in the limb bud formation of vertebrates. Fgf expression in the AER and Shh expression in the ZPA are maintained by their positive feedback regulation mediated by diffusible molecules, FGF and SHH. A recent experimental observation suggests that the FGF-signal regulates the Shh expression in a feed-forward manner with activation and repression regulatory pathways. We study the coupled dynamics of Shh expression in the ZPA and Fgf expression in the AER, and the relationship of the relative position between AER and ZPA. We first show that with the feed-forward regulation only, the peak of ZPA activity can be formed distant from the AER as observed experimentally. Then, we clarify that the robustness of the ZPA spatial pattern to changes in system parameters is enhanced by adding the feedback regulation between the AER and the ZPA. Furthermore, sensitivity analysis shows that there exists the optimal feedback strength where the robustness is the most improved.     

GDF-15 Is Elevated in Children with Mitochondrial Diseases and Is Induced by Mitochondrial Dysfunction

BackgroundWe previously described increased levels of growth and differentiation factor 15 (GDF-15) in skeletal muscle and serum of patients with mitochondrial diseases. Here we evaluated GDF-15 as a biomarker for mitochondrial diseases affecting children and compared it to fibroblast-growth factor 21 (FGF-21). To investigate the mechanism of GDF-15 induction in these pathologies we measured its expression and secretion in response to mitochondrial dysfunction.MethodsWe analysed 59 serum samples from 48 children with mitochondrial disease, 19 samples from children with other neuromuscular diseases and 33 samples from aged-matched healthy children. GDF-15 and FGF-21 circulating levels were determined by ELISA.ResultsOur results showed that in children with mitochondrial diseases GDF-15 levels were on average increased by 11-fold (mean 4046pg/ml, 1492 SEM) relative to healthy (350, 21) and myopathic (350, 32) controls. The area under the curve for the receiver-operating-characteristic curve for GDF-15 was 0.82 indicating that it has a good discriminatory power. The overall sensitivity and specificity of GDF-15 for a cut-off value of 550pg/mL was 67.8% (54.4%-79.4%) and 92.3% (81.5%-97.9%), respectively. We found that elevated levels of GDF-15 and or FGF-21 correctly identified a larger proportion of patients than elevated levels of GDF-15 or FGF-21 alone. GDF-15, as well as FGF-21, mRNA expression and protein secretion, were significantly induced after treatment of myotubes with oligomycin and that levels of expression of both factors significantly correlated.ConclusionsOur data indicate that GDF-15 is a valuable serum quantitative biomarker for the diagnosis of mitochondrial diseases in children and that measurement of both GDF-15 and FGF-21 improves the disease detection ability of either factor separately. Finally, we demonstrate for the first time that GDF-15 is produced by skeletal muscle cells in response to mitochondrial dysfunction and that its levels correlate in vitro with FGF-21 levels.     

Extracellular Glutamate Is Increased in Thalamus During Thiamine Deficiency-Induced Lesions and Is Blocked by MK-801

The current study measured extracellular fluid (ECF) levels of excitatory amino acids before and during the onset of thiamine deficiency-induced pathologic lesions. Male Sprague-Dawley rats were treated with daily pyrithiamine (0.25 mg/kg i.p.) and a thiamine-deficient diet (PTD). Microdialysates were simultaneously collected from probes inserted acutely via guide cannulae into right paracentral and ventrolateral nuclei of thalamus and left hippocampus of PTD and pair-fed controls. Hourly samples were collected from unanesthetized and freely moving animals. Basal levels obtained at a prelesion stage (day 12 of PTD treatment) were unchanged from levels in pairfed controls. In samples collected 4–5 h after onset of seizures (day 14 of PTD), the levels of glutamate were elevated an average 640% of basal levels in medial thalamus and 200% in hippocampus. Glutamine levels declined, taurine and glycine were elevated, and aspartate, GABA, and alanine were unchanged during this period. Within 7 h after seizure onset glutamine was undetectable in both areas, whereas glutamate had declined to ～200% in thalamus and 70% in hippocampus. No significant change in glutamate, aspartate, or other amino acids was observed in dialysates collected from probes located in undamaged dorsal-lateral regions of thalamus. Number of neurons within ventrolateral nucleus of thalamus was significantly greater in PTD animals in which the probe was dialyzed compared with nondialyzed, suggesting that removal of excitatory amino acids was protective. No significant pathologic damage was evident in hippocampus. Pretreatment with MK-801 completely blocked the rise of ECF glutamate and significantly reduced the pathologic damage within thalamus of PTD rats and produced a significant decrease in ECF glutamate in control rats.     

Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation

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Mouse Visual Cortex Is Modulated by Distance Traveled and by Theta Oscillations

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The Ortholog Conjecture Is Untestable by the Current Gene Ontology but Is Supported by RNA Sequencing Data

The ortholog conjecture posits that orthologous genes are functionally more similar than paralogous genes. This conjecture is a cornerstone of phylogenomics and is used daily by both computational and experimental biologists in predicting, interpreting, and understanding gene functions. A recent study, however, challenged the ortholog conjecture on the basis of experimentally derived Gene Ontology (GO) annotations and microarray gene expression data in human and mouse. It instead proposed that the functional similarity of homologous genes is primarily determined by the cellular context in which the genes act, explaining why a greater functional similarity of (within-species) paralogs than (between-species) orthologs was observed. Here we show that GO-based functional similarity between human and mouse orthologs, relative to that between paralogs, has been increasing in the last five years. Further, compared with paralogs, orthologs are less likely to be included in the same study, causing an underestimation in their functional similarity. A close examination of functional studies of homologs with identical protein sequences reveals experimental biases, annotation errors, and homology-based functional inferences that are labeled in GO as experimental. These problems and the temporary nature of the GO-based finding make the current GO inappropriate for testing the ortholog conjecture. RNA sequencing (RNA-Seq) is known to be superior to microarray for comparing the expressions of different genes or in different species. Our analysis of a large RNA-Seq dataset of multiple tissues from eight mammals and the chicken shows that the expression similarity between orthologs is significantly higher than that between within-species paralogs, supporting the ortholog conjecture and refuting the cellular context hypothesis for gene expression. We conclude that the ortholog conjecture remains largely valid to the extent that it has been tested, but further scrutiny using more and better functional data is needed.     

Low Bone Strength Is a Manifestation of Phenylketonuria in Mice and Is Attenuated by a Glycomacropeptide Diet

Purpose

Phenylketonuria (PKU), caused by phenylalanine (phe) hydroxylase loss of function mutations, requires a low-phe diet plus amino acid (AA) formula to prevent cognitive impairment. Glycomacropeptide (GMP), a low-phe whey protein, provides a palatable alternative to AA formula. Skeletal fragility is a poorly understood chronic complication of PKU. We sought to characterize the impact of the PKU genotype and dietary protein source on bone biomechanics.

Procedures

Wild type (WT; Pah^+/+) and PKU (Pah^enu2/enu2) mice on a C57BL/6J background were fed high-phe casein, low-phe AA, and low-phe GMP diets between 3 to 23 weeks of age. Following euthanasia, femur biomechanics were assessed by 3-point bending and femoral diaphyseal structure was determined. Femoral ex vivo bone mineral density (BMD) was assessed by dual-enengy x-ray absorptiometry. Whole bone parameters were used in prinicipal component analysis. Data were analyzed by 3-way ANCOVA with genotype, sex, and diet as the main factors.

Findings

Regardless of diet and sex, PKU femora were more brittle, as manifested by lower post-yield displacement, weaker, as manifested by lower energy and yield and maximal loads, and showed reduced BMD compared with WT femora. Four principal components accounted for 87% of the variance and all differed significantly by genotype. Regardless of genotype and sex, the AA diet reduced femoral cross-sectional area and consequent maximal load compared with the GMP diet.

Conclusions

Skeletal fragility, as reflected in brittle and weak femora, is an inherent feature of PKU. This PKU bone phenotype is attenuated by a GMP diet compared with an AA diet.     

Human Topoisomerase IIIα Is a Single-stranded DNA Decatenase That Is Stimulated by BLM and RMI1

Human topoisomerase IIIα is a type IA DNA topoisomerase that functions with BLM and RMI1 to resolve DNA replication and recombination intermediates. BLM, human topoisomerase IIIα, and RMI1 catalyze the dissolution of double Holliday junctions into noncrossover products via a strand-passage mechanism. We generated single-stranded catenanes that resemble the proposed dissolution intermediate recognized by human topoisomerase IIIα. We demonstrate that human topoisomerase IIIα is a single-stranded DNA decatenase that is specifically stimulated by the BLM-RMI1 pair. In addition, RMI1 interacts with human topoisomerase IIIα, and the interaction is required for the stimulatory effect of RMI1 on decatenase activity. Our data provide direct evidence that human topoisomerase IIIα functions as a decatenase with the assistance of BLM and RMI1 to facilitate the processing of homologous recombination intermediates without crossing over as a mechanism to preserve genome integrity.     

ZIP8 Is an Iron and Zinc Transporter Whose Cell-surface Expression Is Up-regulated by Cellular Iron Loading

ZIP8 (SLC39A8) belongs to the ZIP family of metal-ion transporters. Among the ZIP proteins, ZIP8 is most closely related to ZIP14, which can transport iron, zinc, manganese, and cadmium. Here we investigated the iron transport ability of ZIP8, its subcellular localization, pH dependence, and regulation by iron. Transfection of HEK 293T cells with ZIP8 cDNA enhanced the uptake of ⁵⁹Fe and ⁶⁵Zn by 200 and 40%, respectively, compared with controls. Excess iron inhibited the uptake of zinc and vice versa. In RNA-injected Xenopus oocytes, ZIP8-mediated ⁵⁵Fe²⁺ transport was saturable (K_0.5 of ∼0.7 μm) and inhibited by zinc. ZIP8 also mediated the uptake of ¹⁰⁹Cd²⁺, ⁵⁷Co²⁺, ⁶⁵Zn²⁺ > ⁵⁴Mn²⁺, but not ⁶⁴Cu (I or II). By using immunofluorescence analysis, we found that ZIP8 expressed in HEK 293T cells localized to the plasma membrane and partially in early endosomes. Iron loading increased total and cell-surface levels of ZIP8 in H4IIE rat hepatoma cells. We also determined by using site-directed mutagenesis that asparagine residues 40, 88, and 96 of rat ZIP8 are glycosylated and that N-glycosylation is not required for iron or zinc transport. Analysis of 20 different human tissues revealed abundant ZIP8 expression in lung and placenta and showed that its expression profile differs markedly from ZIP14, suggesting nonredundant functions. Suppression of endogenous ZIP8 expression in BeWo cells, a placental cell line, reduced iron uptake by ∼40%, suggesting that ZIP8 participates in placental iron transport. Collectively, these data identify ZIP8 as an iron transport protein that may function in iron metabolism.     

Hexokinase II Binding to Mitochondria Is Necessary for Kupffer Cell Activation and Is Potentiated by Ethanol Exposure

Ethanol exposure promotes the development of steatohepatitis, which can progress to end stage liver disease. Kupffer cells have been documented to play a key role in the genesis and progression of alcoholic liver disease with ethanol exposure enhancing Kupffer cell activation. In the present study, we identified the binding of hexokinase II to the mitochondria as a requirement for LPS-induced activation of Kupffer cells and its potentiation by ethanol. LPS and ethanol exposure induced a reduction in sirtuin-3 activity. In turn, the decline of sirtuin-3 activity led to the activation of cyclophilin-D, which mediated an increased binding of hexokinase II to the mitochondria. Suppression of cyclophilin-D expression or enforced detachment of hexokinase II from the mitochondria abrogated the LPS- and ethanol-induced stimulation of Kupffer cells, preventing NADPH oxidase and inflammasome activation. Moreover, activation of AMP-activated protein kinase restored sirtuin-3 activity, thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation.     

DNAJB3/HSP-40 Cochaperone Is Downregulated in Obese Humans and Is Restored by Physical Exercise

Obesity is a major risk factor for a myriad of disorders such as insulin resistance and diabetes. The mechanisms underlying these chronic conditions are complex but low grade inflammation and alteration of the endogenous stress defense system are well established. Previous studies indicated that impairment of HSP-25 and HSP-72 was linked to obesity, insulin resistance and diabetes in humans and animals while their induction was associated with improved clinical outcomes. In an attempt to identify additional components of the heat shock response that may be dysregulated by obesity, we used the RT²-Profiler PCR heat shock array, complemented with RT-PCR and validated by Western blot and immunohistochemistry. Using adipose tissue biopsies and PBMC of non-diabetic lean and obese subjects, we report the downregulation of DNAJB3 cochaperone mRNA and protein in obese that negatively correlated with percent body fat (P = 0.0001), triglycerides (P = 0.035) and the inflammatory chemokines IP-10 and RANTES (P = 0.036 and P = 0.02, respectively). DNAJB positively correlated with maximum oxygen consumption (P = 0.031). Based on the beneficial effect of physical exercise, we investigated its possible impact on DNAJB3 expression and indeed, we found that exercise restored the expression of DNAJB3 in obese subjects with a concomitant decrease of phosphorylated JNK. Using cell lines, DNAJB3 protein was reduced following treatment with palmitate and tunicamycin which is suggestive of the link between the expression of DNAJB3 and the activation of the endoplasmic reticulum stress. DNAJB3 was also shown to coimmunoprecipiate with JNK and IKKβ stress kinases along with HSP-72 and thus, suggesting its potential role in modulating their activities. Taken together, these data suggest that DNAJB3 can potentially play a protective role against obesity.     

Cardiac Autonomic Modulation Is Determined by Gender and Is Independent of Aerobic Physical Capacity in Healthy Subjects

Background

Aerobic physical capacity plays an important role in reducing morbidity and mortality rates in subjects with cardiovascular diseases. This action is often related to an improvement in the autonomic modulation of heart rate variability (HRV). However, controversies remain regarding the effects of physical training on cardiac autonomic control in healthy subjects. Therefore, our objective was to investigate whether aerobic capacity interferes with the autonomic modulation of HRV and whether gender differences exist.

Methods

Healthy men and women (N=96) were divided into groups according to aerobic capacity: low (VO₂: 22-38 ml/kg^-1 min^-1), moderate (VO₂: 38-48 ml/kg^-1 min^-1) and high (VO₂ >48 ml/kg^-1 min^-1.) We evaluated the hemodynamic parameters and body composition. The autonomic modulation of HRV was investigated using spectral analysis. This procedure decomposes the heart rate oscillatory signal into frequency bands: low frequency (LF=0.04-0.15Hz) is mainly related to sympathetic modulation, and high frequency (HF=0.15-0.5Hz) corresponds to vagal modulation.

Results

Aerobic capacity, regardless of gender, determined lower values of body fat percentage, blood pressure and heart rate. In turn, the spectral analysis of HRV showed that this parameter did not differ when aerobic capacity was considered. However, when the genders were compared, women had lower LF values and higher HF values than the respective groups of men.

Conclusion

The results suggest that aerobic physical capacity does not interfere with HRV modulation; however, the cardiac modulatory balance differs between genders and is characterized by a greater influence of the autonomic vagal component in women and by the sympathetic component in men.     

Serum IgE Induced Airway Smooth Muscle Cell Remodeling Is Independent of Allergens and Is Prevented by Omalizumab

Background

Airway wall remodeling in allergic asthma is reduced after treatment with humanized anti-IgE-antibodies. We reported earlier that purified IgE, without the presence of allergens, is sufficient to induce airway wall remodeling due to airway smooth muscle cell (ASMC) activity deposing extracellular matrix.

Objective

We postulate that IgE contained in serum of allergic asthma patients, in the absence of allergens, stimulates ASMC remodeling activities and can be prevented by anti-IgE antibodies.

Methods

Isolated human ASMC were exposed to serum obtained from: (i) healthy controls, or patients with (ii) allergic asthma, (iii) non-allergic asthma, and (iv) atopic non-asthma patients. Proliferation and the deposition of collagens and fibronectin were determined after 3 and 5 days.

Results

Serum from patients with allergies significantly stimulated: (i) ASMC proliferation, (ii) deposition of collagen type-I (48 hours) and (iii) of fibronectin (24 hours). One hour pre-incubation with Omalizumab prevented these three effects of allergic serum, but had no significant effect on serum from healthy donors or non-allergic asthma patients. Interestingly, the addition of allergens did not further increase any of the IgE effects.

Conclusion and Clinical Relevance

Our data provides experimental evidence that the beneficial effect of Omalizumab on airway wall remodeling and improved lung function may be due to its direct action on IgE bound ASMC.     

Vertebrate Decomposition Is Accelerated by Soil Microbes

Carrion decomposition is an ecologically important natural phenomenon influenced by a complex set of factors, including temperature, moisture, and the activity of microorganisms, invertebrates, and scavengers. The role of soil microbes as decomposers in this process is essential but not well understood and represents a knowledge gap in carrion ecology. To better define the role and sources of microbes in carrion decomposition, lab-reared mice were decomposed on either (i) soil with an intact microbial community or (ii) soil that was sterilized. We characterized the microbial community (16S rRNA gene for bacteria and archaea, and the 18S rRNA gene for fungi and microbial eukaryotes) for three body sites along with the underlying soil (i.e., gravesoils) at time intervals coinciding with visible changes in carrion morphology. Our results indicate that mice placed on soil with intact microbial communities reach advanced stages of decomposition 2 to 3 times faster than those placed on sterile soil. Microbial communities associated with skin and gravesoils of carrion in stages of active and advanced decay were significantly different between soil types (sterile versus untreated), suggesting that substrates on which carrion decompose may partially determine the microbial decomposer community. However, the source of the decomposer community (soil- versus carcass-associated microbes) was not clear in our data set, suggesting that greater sequencing depth needs to be employed to identify the origin of the decomposer communities in carrion decomposition. Overall, our data show that soil microbial communities have a significant impact on the rate at which carrion decomposes and have important implications for understanding carrion ecology.     

Chromosome Segregation Is Biased by Kinetochore Size

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