Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis.

Methods

The expression of TLR-2, TLR-4 and CD14 in monocytes was analyzed by flow cytometry. To study the functional responses of these receptors, monocytes were stimulated with peptidoglycan or lipopolysaccharide and the amounts of TNFα and IL-6 secreted were determined by ELISA.

Results

We found that the expression of TLR-2 was up-regulated in peripheral blood monocytes from COPD patients, either clinically stable or during AECOPD, as compared to never smokers or smokers with normal lung function. Upon stimulation with TLR-2 ligand monocytes from COPD patients secreted increased amounts of cytokines than similarly stimulated monocytes from never smokers and smokers. In contrast, the expressions of TLR-4 and CD14 were not significantly different between groups, and the response to lipopolysaccharide (a TLR-4 ligand) stimulation was not significantly different either. At discharge from hospital TLR-2 expression was down-regulated in peripheral blood monocytes from AECOPD patients. This could be due to the treatment with systemic steroids because, in vitro, steroids down-regulated TLR-2 expression in a dose-dependent manner. Finally, we demonstrated that IL-6, whose plasma levels are elevated in patients, up-regulated in vitro TLR-2 expression in monocytes from never smokers.

Conclusion

Our results reveal abnormalities in TLRs expression in COPD patients and highlight its potential relationship with systemic inflammation in these patients.

     

Analysis of the role of the leucine zipper motif in regulating the ability of AFAP-110 to alter actin filament integrity

AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.     

A potential mechanism for cryptic female choice in a flour beetle

In polyandrous mating systems, events occurring during copulation can alter the fate of the mating male's sperm. These events may exert selective pressures resulting in the evolution of diverse reproductive behaviours, morphologies and physiologies. This study investigates the role of male and female copulatory behaviours on sperm fate in the red flour beetle, Tribolium castaneum. I describe and quantify copulatory behaviours for mating pairs, and examine sperm fate by quantifying sperm transfer, sperm storage and second male sperm precedence. Whereas female quiescence during copulation and male leg rubbing during copulation together account for significant variation (26%) in sperm precedence, female copulatory quiescence alone provides information about the timing and numbers of sperm transferred. These experiments show that a female copulatory behaviour predicts sperm fate, and emphasize the value of studying variation in both male and female copulatory behaviours for understanding factors affecting sperm use.     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Phosphorylation of Bcl-2 and Bax Proteins during Hypoxia in Newborn Piglets

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca²⁺ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Development and Internal Validation of a Predictive Model Including Pulse Oximetry for Hospitalization of Under-Five Children in Bangladesh

Background

The reduction in the deaths of millions of children who die from infectious diseases requires early initiation of treatment and improved access to care available in health facilities. A major challenge is the lack of objective evidence to guide front line health workers in the community to recognize critical illness in children earlier in their course.

Methods

We undertook a prospective observational study of children less than 5 years of age presenting at the outpatient or emergency department of a rural tertiary care hospital between October 2012 and April 2013. Study physicians collected clinical signs and symptoms from the facility records, and with a mobile application performed recordings of oxygen saturation, heart rate and respiratory rate. Facility physicians decided the need for hospital admission without knowledge of the oxygen saturation. Multiple logistic predictive models were tested.

Findings

Twenty-five percent of the 3374 assessed children, with a median (interquartile range) age of 1.02 (0.42–2.24), were admitted to hospital. We were unable to contact 20% of subjects after their visit. A logistic regression model using continuous oxygen saturation, respiratory rate, temperature and age combined with dichotomous signs of chest indrawing, lethargy, irritability and symptoms of cough, diarrhea and fast or difficult breathing predicted admission to hospital with an area under the receiver operating characteristic curve of 0.89 (95% confidence interval -CI: 0.87 to 0.90). At a risk threshold of 25% for admission, the sensitivity was 77% (95% CI: 74% to 80%), specificity was 87% (95% CI: 86% to 88%), positive predictive value was 70% (95% CI: 67% to 73%) and negative predictive value was 91% (95% CI: 90% to 92%).

Conclusion

A model using oxygen saturation, respiratory rate and temperature in combination with readily obtained clinical signs and symptoms predicted the need for hospitalization of critically ill children. External validation of this model in a community setting will be required before adoption into clinical practice.     

Interaction pattern for the complex of B-DNA Fullerene compounds with a set of known replication proteins using docking study

Fullerenes have attracted considerable attention due to their unique chemical structure and potential applications which has opened wide venues for possible human exposure to various fullerene types. Therefore, in depth knowledge of how fullerene may interfere with various cellular processes becomes quite imperative. The present study was designed to investigate how the presence of fullerene affect the binding of DNA with different enzymes involved in replication process. Different fullerenes were first docked with DNA and then binding scores of different enzymes was analyzed with fullerene docked DNA. C30, C40 & C50 once docked with DNA, reduced the binding score of primase, whereas no significant change in the binding score was observed with the helicase, ssb protein, dna pol δ, dna pol ε, ligase, DNA clamp, and topoisomerases. On the contrast, the binding score of RPA14 decreases in fluctuating manner while interacting with increasing molecular weight of fullerene bound single-stranded DNA complex. The study revealed the affect of fullerene family interacting with DNA on the binding pattern of enzymes involved in replication process. Study suggests that the presence of most of fullerenes may not affect the activity of these enzymes necessary for replication process whereas C30, C40 & C50 may disrupt the activity of primase, (strating point for DNA polymerase) its docking score decreases from 13820 to 10702.     

Salt-induced modulation in growth,photosynthesis and antioxidant system in two varieties of Brassica juncea

The present study was carried out to examine salt-induced modulation in growth, photosynthetic characteristics and antioxidant system in two cultivars of Brassica juncea Czern and Coss varieties (Varuna and RH-30). The surface sterilized seeds of these varieties were sown in the soil amended with different levels (2.8, 4.2 or 5.6 dsm⁻¹) of sodium chloride under a simple randomized block design. The salt treatment significantly decreased growth, net photosynthetic rate and its related attributes, chlorophyll fluorescence, SPAD value of chlorophyll, leaf carbonic anhydrase activity and leaf water potential, whereas electrolyte leakage, proline content, and activity of catalase, peroxidase and superoxide dismutase enzymes increased in both the varieties at 30 d stage of growth. The variety Varuna was found more resistant than RH-30 to the salt stress and possessed higher values for growth, photosynthetic attributes and antioxidant enzymes. Out of the graded concentrations (2.8, 4.2 or 5.6 dsm⁻¹) of sodium chloride, 2.8 sm⁻¹ was least toxic and 5.6 dsm⁻¹ was most harmful. The variation in the responses of these two varieties to salt stress is attributed to their differential photosynthetic traits, SPAD chlorophyll value and antioxidant capacity, which can be used as potential markers for screening mustard plants for salt tolerance.     

Lipase-catalyzed regioselective protection/deprotection of hydroxyl groups of the isoflavone irilone isolated from Iris germanica

The regioselective acylation of irilone, isolated from Iris germanica, with vinylacetate and propenylacetate and deacylation of irilone diacetate with n-butanol were studied using lipases from Aspergillus niger, Mucor miehei, Pseudomonas cepacia, Candida cylindracea, porcine pancreas and Candida antarctica. Significant conversion of irilone to 4′-O-acetylirilone was achieved using P. cepacia lipase, while irilone diacetate was converted to 5-O-acetylirilone by the enzymatic action of lipases from M. miechei, P. cepacia and porcine pancreas under different experimental conditions. This preferential protection/deprotection furnishes an opportunity to modify the structure of irilone by selective derivatization that may help to change its biological activities by modifying its amphiphilic/lipophilic balance.