Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle

Muscle contractions strongly activate p38 MAP kinases, but the precise contraction‐associated sarcoplasmic event(s) (e.g., force production, energetic demands, and/or calcium cycling) that activate these kinases are still unclear. We tested the hypothesis that during contraction the phosphorylation of p38 isoforms is sensitive to the increase in ATP demand relative to ATP supply. Energetic demands were inhibited using N‐benzyl‐p‐toluene sulphonamide (BTS, type II actomyosin) and cyclopiazonic acid (CPA, SERCA). Extensor digitorum longus muscles from Swiss Webster mice were incubated in Ringer's solution (37°C) with or without inhibitors and then stimulated at 10 Hz for 15 min. Muscles were immediately freeze‐clamped for metabolite and Western blot analysis. BTS and BTS + CPA treatment decreased force production by 85%, as measured by the tension time integral, while CPA alone potentiated force by 310%. In control muscles, contractions resulted in a 73% loss of ATP content and a concomitant sevenfold increase in IMP content, a measure of sustained energetic imbalance. BTS or CPA treatment lessened the loss of ATP, but BTS + CPA treatment completely eliminated the energetic imbalance since ATP and IMP levels were nearly equal to those of non‐stimulated muscles. The independent inhibition of cytosolic ATPase activities had no effect on contraction‐induced p38 MAPK phosphorylation, but combined treatment prevented the increase in phosphorylation of the γ isoform while the α/β isoforms unaffected. These observations suggest that an energetic signal may trigger phosphorylation of the p38γ isoform and also may explain how contractions differentially activate signaling pathways. J. Cell. Biochem. 114: 1445–1455, 2013. © 2013 Wiley Periodicals, Inc.     

A perforin‐like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin‐like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore‐forming toxin, we conclude that rupture of the erythrocyte membraneis blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin‐like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Energy deprivation by silibinin in colorectal cancer cells

Small molecules with the potential to initiate different types of programmed cell death could be useful ‘adjunct therapy’ where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨ_m) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT–MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly ‘double-edged sword’ against CRC cells thereby further advocating its clinical effectiveness against this malignancy.     

Findings from the Peutz-Jeghers Syndrome Registry of Uruguay

Background

Peutz-Jeghers syndrome (PJS) is characterized by intestinal polyposis, mucocutaneous pigmentation and an increased cancer risk, usually caused by mutations of the STK11 gene. This study collected epidemiological, clinical and genetic data from all Uruguayan PJS patients.

Methods

Clinical data were obtained from public and private medical centers and updated annually. Sequencing of the STK11 gene in one member of each family was performed.

Results and discussion

25 cases in 11 unrelated families were registered (15 males, 10 females). The average age of diagnosis and death was 18 and 41 years respectively. All patients had characteristic PJS pigmentation and gastrointestinal polyps. 72% required urgent surgery due to intestinal obstruction. 3 families had multiple cases of seizure disorder, representing 20% of cases. 28% developed cancer and two patients had more than one cancer. An STK11 mutation was found in 8 of the 9 families analyzed. A unique M136K missense mutation was noted in one family. Comparing annual live births and PJS birth records from 1970 to 2009 yielded an incidence of 1 in 155,000.

Conclusion

The Uruguayan Registry for Peutz-Jeghers patients showed a high chance of emergent surgery, epilepsy, cancer and shortened life expectancy. The M136K missense mutation is a newly reported STK 11 mutation.     

Human Circulating Antibody-Producing B Cell as a Predictive Measure of Mucosal Immunity to Poliovirus

Background

The “gold standard” for assessing mucosal immunity after vaccination with poliovirus vaccines consists in measuring virus excretion in stool after challenge with oral poliovirus vaccine (OPV). This testing is time and resource intensive, and development of alternative methods is a priority for accelerating polio eradication. We therefore evaluated circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immunity to poliovirus vaccine.

Methods

199 subjects, aged 10 years, and previously immunized repeatedly with OPV, were selected. Subjects were assigned to receive either a booster dose of inactivated poliovirus vaccine (IPV), bivalent OPV (bOPV), or no vaccine. Using a micro-modified whole blood-based ELISPOT assay designed for field setting, circulating poliovirus type-specific IgA- and IgG-ASCs, including gut homing α4β7+ ASCs, were enumerated on days 0 and 7 after booster immunization. In addition, serum samples collected on days 0, 28 and 56 were tested for neutralizing antibody titers against poliovirus types 1, 2, and 3. Stool specimens were collected on day 28 (day of bOPV challenge), and on days 31, 35 and 42 and processed for poliovirus isolation.

Results

An IPV dose elicited blood IgA- and IgG-ASC responses in 84.8 to 94.9% of subjects, respectively. In comparison, a bOPV dose evoked corresponding blood ASC responses in 20.0 to 48.6% of subjects. A significant association was found between IgA- and IgG-ASC responses and serum neutralizing antibody titers for poliovirus type 1, 2, 3 (p<0.001). In the IPV group, α4β7⁺ ASCs accounted for a substantial proportion of IgA-ASCs and the proportion of subjects with a positive α4β7⁺ IgA-ASC response to poliovirus types 1, 2 and 3 was 62.7%, 89.8% and 45.8%, respectively. A significant association was observed between virus excretion and α4β7⁺ IgA^- and/or IgG-ASC responses to poliovirus type 3 among immunized children; however, only a weak association was found for type 1 poliovirus.

Discussion

Our results suggest that virus-specific blood ASCs, especially for type 3 poliovirus, can serve as surrogate of mucosal immunity after vaccination. Further studies are needed to evaluate the duration of such memory responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the polio eradication program.     

Can habitat characteristics shape vertebral morphology in dolphins? An example of two phylogenetically related species from southern South America

Fast swimming pelagic cetacean species have osteological characteristics that promote a more stable spine in comparison to that of coastal species. The Peale's dolphin (Lagenorhynchus australis) and the hourglass dolphin (Lagenorhynchus cruciger) have a close phylogenetic relationship and are found in coastal and pelagic waters in the Southern Hemisphere, respectively. The aim of this work was to study the relationship between the vertebral column's morphology and its flexibility, across these species of contrasting habitats. Vertebral counts and multiple measurements of each vertebra were used to infer intervertebral flexibility. Bivariate plots and discriminant multivariate analyses were employed to compare each functional region along the vertebral column. Both species displayed a regionalization of the column into three stable regions and two flexible areas, which statistically differ in the proportion of the skeleton occupied in each species. While the Peale's dolphin has rounder vertebrae, associated with higher flexibility, the hourglass dolphin has disk‐shaped vertebrae and strongly inclined processes related to high stability. Although the species are closely related phylogenetically, vertebral morphology is influenced by a diverse set of ecological and behavioral factors, reflecting a high degree of vertebral plasticity within the genus.     

Critical determinants of the interactions of capsule-expressing Neisseria meningitidis with host cells: the role of receptor density in increased cellular targeting via the outer membrane Opa proteins

Neisseria meningitidis capsule is an important virulence determinant required for survival in the blood but is reportedly involved in inhibiting cellular interactions mediated by meningococcal outer membrane adhesins. However, evidence from our previous studies suggested that target receptor density on host cells may determine whether or not capsulate bacteria can adhere via outer membrane proteins such as Opa. To confirm this and evaluate the impact of capsulation on bacterial interactions, we used Opa(+) and Opa(-) derivatives of capsulate and acapsulate meningococcal isolates and transfected cell lines expressing CEACAM1, a receptor targeted by Opa proteins. To assess the extent and rate of cell association, subpopulations of stably transfected Chinese hamster ovary cells with different receptor levels were derived. A quantitative correlation of CEACAM1 levels and Opa-dependent binding of both capsulate and acapsulate bacteria was demonstrated, which was accelerated at high receptor densities. However, it appears that invasion by Opa(+) capsulate bacteria only occurs when a threshold level of CEACAM density has been reached. Target cells expressing high levels of CEACAM1 (MFI c. 400) bound threefold more, but internalized 20-fold more Opa(+) capsulate bacteria than those with intermediate expression (MFI c. 100). No overall selection of acapsulate phenotype was observed in the internalized population. These observations confirm that capsule may not be an adequate barrier for cellular interactions and demonstrate the role of a host factor that may determine capsulate bacterial invasion potential. Upregulation of CEACAMs, which can occur in response to inflammatory cytokines, could lead to translocation of a small number of fully capsulate bacteria across mucosal epithelium into the bloodstream sufficient to cause a rapid onset of disseminated disease. Thus the data also suggest a novel rationale for the epidemiological observations that individuals with prior infectious/inflammatory conditions carry a high risk of invasive meningococcal disease.