Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

On the accuracy of the Head Impact Telemetry (HIT) System used in football helmets

On-field measurement of head impacts has relied on the Head Impact Telemetry (HIT) System, which uses helmet mounted accelerometers to determine linear and angular head accelerations. HIT is used in youth and collegiate football to assess the frequency and severity of helmet impacts. This paper evaluates the accuracy of HIT for individual head impacts. Most HIT validations used a medium helmet on a Hybrid III head. However, the appropriate helmet is large based on the Hybrid III head circumference (58 cm) and manufacturer's fitting instructions. An instrumented skull cap was used to measure the pressure between the head of football players (n=63) and their helmet. The average pressure with a large helmet on the Hybrid III was comparable to the average pressure from helmets used by players. A medium helmet on the Hybrid III produced average pressures greater than the 99th percentile volunteer pressure level. Linear impactor tests were conducted using a large and medium helmet on the Hybrid III. Testing was conducted by two independent laboratories. HIT data were compared to data from the Hybrid III equipped with a 3-2-2-2 accelerometer array. The absolute and root mean square error (RMSE) for HIT were computed for each impact (n=90). Fifty-five percent (n=49) had an absolute error greater than 15% while the RMSE was 59.1% for peak linear acceleration.     

Undiagnosed Cryptic Diversity in Small,Microendemic Frogs (Leptolalax) from the Central Highlands of Vietnam

A major obstacle in prioritizing species or habitats for conservation is the degree of unrecognized diversity hidden within complexes of morphologically similar, “cryptic” species. Given that amphibians are one of the most threatened groups of organisms on the planet, our inability to diagnose their true diversity is likely to have significant conservation consequences. This is particularly true in areas undergoing rapid deforestation, such as Southeast Asia. The Southeast Asian genus Leptolalax is a group of small-bodied, morphologically conserved frogs that inhabit the forest-floor. We examined a particularly small-bodied and morphologically conserved subset, the Leptolalax applebyi group, using a combination of molecular, morphometric, and acoustic data to identify previously unknown diversity within. In order to predict the geographic distribution of the group, estimate the effects of habitat loss and assess the degree of habitat protection, we used our locality data to perform ecological niche modelling using MaxEnt. Molecular (mtDNA and nuDNA), acoustic and subtle morphometric differences revealed a significant underestimation of diversity in the L. applebyi group; at least two-thirds of the diversity may be unrecognised. Patterns of diversification and microendemism in the group appear driven by limited dispersal, likely due to their small body size, with several lineages restricted to watershed basins. The L. applebyi group is predicted to have historically occurred over a large area of the Central Highlands of Vietnam, a considerable portion of which has already been deforested. Less than a quarter of the remaining forest predicted to be suitable for the group falls within current protected areas. The predicted distribution of the L. applebyi group extends into unsurveyed watershed basins, each potentially containing unsampled diversity, some of which may have already been lost due to deforestation. Current estimates of amphibian diversity based on morphology alone are misleading, and accurate alpha taxonomy is essential to accurately prioritize conservation efforts.     

Comparison of light induced and cell cycle dependent changes in the photosynthetic apparatus: A fluorescence induction study on the green alga Scenedesmus obliquus

Changes in the photosynthetic apparatus occurring during the synchronous cell cycle of the green alga Scenedesmus obliquus are compared to the adaptational response induced by light intensity variations. To investigate and compare these two phenomena, we analyze the polyphasic rise of the chlorophyll fluorescence yield exhibited by plants and cyanobacteria when exposed to high intensity actinic light. Four fluorescence parameters are calculated which are closely related to Photosystem II (PS II) structure and function: ABS/RC, the antenna size of PS II; _PO, the quantum yield for reduction of the primary PS II quinone acceptor; q_PQ, related to the size of the plastoquinone pool; q_Emax, the capacity for pH dependent non-photochemical quenching. The capacity for non-photochemical quenching changes in response to light intensity variations, but it is not affected by the developmental changes occurring during the cell cycle. In contras t, for ABS/RC, _PO and q_PQ, we observe light induced as well as cell cycle dependent variations. We discuss the relations of the four fluorescence parameters to the molecular organization of the photosynthetic apparatus and its cell cycle and light dependent changes.     

Adaptation of the Photosynthetic Apparatus of Anacystis nidulans to Irradiance and CO2-Concentration

Homocontinuous cultures of the cyanobacterium Anacystis nidulans (syn. Synechococcus sp. PCC 6301) were grown at white light intensities of 2 and 20 W/m², and supplied with 0.03 and 3 % CO₂ enriched air. The mutual influence of these growth factors on the development of the photosynthetic apparatus was studied by analyses of the pigment content, by low temperature absorbance and fluorescence spectroscopy, by analyses of oxygen evolution light-saturation curves, and by SDS PAGE of isolated phycobilisomes. The two growth factors, light and CO₂, distinctly affect the absorption cross section of the photosynthetic apparatus, which is expressed by its pigment pattern, excitation energy distribution and capacity. In response to low CO₂ concentrations, the phycocyanin / allophycocyanin ratios were lower and one linker polypeptide L³⁰_R, of the phycobilisomes was no longer detectable in SDS PAGE. Apparently, low CO₂ adaptation results in shorter phycobilisome rods. Specifically, upon adaptation to low light intensities, the chlorophyll and the phycocyanin content on a per cell basis increase by about 50% suggesting a parallel increase in the amount of phycobilisomes and photosystem core-complexes. Low light adaptation and low CO₂ adaptation both cause a shift of the excitation energy distribution in favor of photosystem I. Variations in the content of the “anchor” polypeptides L⁶⁰_CM and L⁷⁵_CM are possibly related to changes in the excitation energy transfer from phycobilisomes to the photosystem II and photosystem I core-complexes.     

Studies on the adaptation of intact leaves to changing light intensities by a kinetic analysis of chlorophyll fluorescence and oxygen evolution as measured by the photoacoustic signal

A detailed quantitative study of the kinetics of photochemical and non-photochemical quenching was achieved by a linear analysis of the yields of chlorophyll fluorescence and of oxygen evolution (as measured by the photoacoustic effect) by their responses to sinusoidal changes of actinic light. The results of this analysis were given in terms of the parameters of the kinetic phases obtained as a response to a step function change in the light intensity from a previous steady-state. Thus, it was possible to split the responses to a change in light intensity into six components which could be assigned to 6 time-constants (60 ms, 1.8 s, 2.5 s, 8 s, 150 s and 400 s). The comparison of the kinetics of responses induced by blue-light (approx. 400–500 nm) and by far-red (720 nm) light led to the assignment of the 1.8-s time-constant to the loading and discharge of the plastoquinone pool and of the 400-s time-constant to the state-transition controller which could be shown to be involved also in the adaptation to changes in light intensity and not only to changes in light quality (wavelength). The time-constant of 8 s, also occurring in 532-nm light-scattering was assigned to the high-energy state quenching (q_E) of fluorescence. q_E was paralleled by a decrease of the photoacoustic signal, demonstrating an high-energy state quenching of oxygen evolution as well. The 60-ms time-constant is suggested to be related to the redox state of the primary quinone acceptor of PS II, whereas the other two time-constants could not be identified. The calculation of the relative contributions of the photo-chemical and of the non-photochemical quenching in the individual components revealed that both quenching-mechanisms occur in all components except in the assumed fastest one.Abbreviations pa-signal photoacoustic signal - PS photosystem     

The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193-0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15-2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis.     

Stepwise Transition of the Tetra-Manganese Complex of Photosystem II to a Binuclear Mn2(μ-O)2 Complex in Response to a Temperature Jump: A Time-Resolved Structural Investigation Employing X-Ray Absorption Spectroscopy

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25°C to 47°C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y_D^ox radical and of released Mn²⁺ by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y_D^ox occur simultaneously (k₁ = 1.0 min⁻¹). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by ~3.3 Å, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by ~2.7 Å in the native complex are reduced to Mn(II) and released (k₂ = 0.18 min⁻¹). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di-μ-oxo bridged Mn(III)₂ complex. (3) Finally, the tightly-bound Mn₂(μ-O)₂ unit is slowly reduced and released (k₃ = 0.014 min⁻¹).     

On the structure of the manganese complex of photosystem II: extended-range EXAFS data and specific atomic-resolution models for four S-states

The water-oxidizing manganese complex bound to the proteins of photosystem II (PSII) was studied by X-ray absorption spectroscopy on PSII membrane particles. An extended range for collection of extended X-ray absorption fine-structure (EXAFS) data was used (up to 16.6A(-1)). The EXAFS suggests the presence of two Mn-Mn distances close to 2.7A (per Mn4Ca complex); the existence of a third Mn-Mn distance below 2.9A is at least uncertain. Interestingly, a distance of 3.7A is clearly resolved in the extended-range data and tentatively assigned to a Mn-Mn distance. Taking into account the above EXAFS results (inter alia), we present a model for the structure of the PSII manganese complex, which differs from previous atomic-resolution models. Emphasizing the hypothetical character, we propose for all semi-stable S-states: (i) a structure of the Mn4Ca(mu-O)n core, (ii) a model of the amino acid environment, and (iii) assignments of distinct Mn oxidation states to all the individual Mn ions. This specific working model may permit discussion, verification and invalidation of its various features in comparison with experimental and theoretical findings.     

15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-induced cyclooxygenase-2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel-shift analyses demonstrated that 15d-PGJ2 inhibited NF-kappaB activation, while troglitazone was ineffective. Although 15d-PGJ2 attenuated activator protein-1 binding on the DNA, it potentiated c-jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d-PGJ2 action in human chondrocytes is mainly PPARgamma-independent.