The comparison of peroxynitrite action on bovine, porcine and human fibrinogens

Subjects of bovine and porcine flocks are sometimes susceptible to death before time of slaughter, and some of those deaths may be due to cardiovascular problems connected with stress. The role of oxidative stress in farm animals is yet unexplored. Human fibrinogen seems to be highly susceptible to nitration. Peroxynitrite produced from superoxide and nitric oxide initiates noticeable changes in the structure of human fibrinogen molecule. The objective of this work is to compare the in vitro interactions of peroxynitrite with human fibrinogen and with fibrinogen from mammals of great economic importance, namely cows and pigs. Using western blots and ELISA we show that porcine fibrinogen is susceptible to tyrosine nitration induced by peroxynitrite whereas, bovine fibrinogen is more resistant. Moreover, porcine fibrinogen polymerization is susceptible to peroxynitrite action, whereas bovine fibrinogen is the least susceptible to inhibition of polymerization caused by peroxynitrite. These observed differences may result from differences in amino acid sequence of fibrinogen chains, mostly including tyrosine content and location in the Aα chain. Protection against toxic effects of peroxynitrite activity in the circulatory system seems to be important in avoiding cardiovascular diseases and may prevent production loss in pig breeding herds.     

Relationship between the percentage of body fat and surrogate indices of fatness in male and female Polish active and sedentary students

Background

Limited data have indicated that body mass index (BMI), waist circumference (WC), waist to hip ratio (WHR) and waist to height ratio (WHtR) of athletes and young adults provide misleading results concerning body fat content. This study was aimed at the evaluation of the relationship between different surrogate indices of fatness (BMI, WC, WHR, WHtR and body adiposity index (BAI)) with the percentage of body fat in Polish students with respect to their sex and physical activity.

Methods

A total of 272 students volunteered to participate in the study. Of these students, 177 physical education students (90 males and 87 females) were accepted as active (physical activity of 7 to 9 hours/week); and 95 students of other specializations (49 males and 46 females) were accepted as sedentary (physical activity of 1.5 hours/week). Weight, height, waist and hip circumferences were measured, and BMI, WHR, WHtR and BAI were calculated. Body fat percentage was assessed using four skinfold measurements.

Results

Classification of fatness according to the BMI and the percentage of body fat have indicated that BMI overestimates fatness in lean subjects (active men and women, sedentary men), but underestimates body fat in obese subjects (sedentary women). In all groups, BMI, WHR, WHtR and BAI were significantly correlated with the percentage of body fat (with the exception of WHR and hip circumference in active and sedentary women, respectively). However, coefficients of determination not exceeding 50% and Lin’s concordance correlation coefficients lower than 0.9 indicated no relationship between measured and calculated body fat.

Conclusion

The findings in the present study support the concept that irrespective of physical activity and sex none of the calculated indices of fatness are useful in the determination of body fat in young adults. Thus, it seems that easily calculated indices may contribute to distorted body image and unhealthy dietary habits observed in many young adults in Western countries, but also in female athletes.     

Thermal Nociceptive Threshold Testing Detects Altered Sensory Processing in Broiler Chickens with Spontaneous Lameness

Lameness is common in commercially reared broiler chickens but relationships between lameness and pain (and thus bird welfare) have proved complex, partly because lameness is often partially confounded with factors such as bodyweight, sex and pathology. Thermal nociceptive threshold (TNT) testing explores the neural processing of noxious stimuli, and so can contribute to our understanding of pain. Using an acute model of experimentally induced articular pain, we recently demonstrated that TNT was reduced in lame broiler chickens, and was subsequently attenuated by administration of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). This study extended these findings to a large sample of commercial broilers. It examined factors affecting thermal threshold (Part 1) and the effect of an NSAID drug (meloxicam, 5 mg/kg) and of an opioid (butorphanol; 4 mg/kg) (Part 2). Spontaneously lame and matched non-lame birds (n = 167) from commercial farms were exposed to ramped thermal stimulations via a probe attached to the lateral aspect of the tarsometatarsus. Baseline skin temperature and temperature at which a behavioural avoidance response occurred (threshold) were recorded. In Part 1 bird characteristics influencing threshold were modelled; In Part 2 the effect of subcutaneous administration of meloxicam or butorphanol was investigated. Unexpectedly, after accounting for other influences, lameness increased threshold significantly (Part 1). In Part 2, meloxicam affected threshold differentially: it increased further in lame birds and decreased in non-lame birds. No effect of butorphanol was detected. Baseline skin temperature was also consistently a significant predictor of threshold. Overall, lameness significantly influenced threshold after other bird characteristics were taken into account. This, and a differential effect of meloxicam on lame birds, suggests that nociceptive processing may be altered in lame birds, though mechanisms for this require further investigation.     

Postural Stability in Young Adults with Down Syndrome in Challenging Conditions

To evaluate postural control and performance in subjects with Down syndrome (SwDS), we measured postural sway (COP) in quiet stance in four 20-second tests: with eyes open or closed and on hard or foam surface. Ten SwDS and eleven healthy subjects participated, aged 29.8 (4.8) and 28.4 (3.9), respectively. The time-series recorded with the sampling rate of 100 Hz were used to evaluate postural performance (COP amplitude and mean velocity) and strategies (COP frequency, fractal dimension and entropy). There were no intergroup differences in the amplitude except the stance on foam pad with eyes open when SwDS had larger sway. The COP velocity and frequency were larger in SwDS than controls in all trials on foam pad. During stances on the foam pad SwDS increased fractal dimension showing higher complexity of their equilibrium system, while controls decreased sample entropy exhibiting more conscious control of posture in comparison to the stances on hard support surface. This indicated that each group used entirely different adjustments of postural strategies to the somatosensory challenge. It is proposed that the inferior postural control of SwDS results mainly from insufficient experience in dealing with unpredictable postural stimuli and deficit in motor learning.     

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.     

RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.