Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells

How the pore-forming protein perforin delivers apoptosis-inducing granzymes to the cytosol of target cells is uncertain. Perforin induces a transient Ca2+ flux in the target cell, which triggers a process to repair the damaged cell membrane. As a consequence, both perforin and granzymes are endocytosed into enlarged endosomes called 'gigantosomes'. Here we show that perforin formed pores in the gigantosome membrane, allowing endosomal cargo, including granzymes, to be gradually released. After about 15 min, gigantosomes ruptured, releasing their remaining content. Thus, perforin delivers granzymes by a two-step process that involves first transient pores in the cell membrane that trigger the endocytosis of granzyme and perforin and then pore formation in endosomes to trigger cytosolic release.     

Role for miR-204 in human pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Because microRNAs have been recently implicated in the regulation of cell proliferation and apoptosis, we hypothesized that these regulatory molecules might be implicated in the etiology of PAH. In this study, we show that miR-204 expression in PASMCs is down-regulated in both human and rodent PAH. miR-204 down-regulation correlates with PAH severity and accounts for the proliferative and antiapoptotic phenotypes of PAH-PASMCs. STAT3 activation suppresses miR-204 expression, and miR-204 directly targets SHP2 expression, thereby SHP2 up-regulation, by miR-204 down-regulation, activates the Src kinase and nuclear factor of activated T cells (NFAT). STAT3 also directly induces NFATc2 expression. NFAT and SHP2 were needed to sustain PAH-PASMC proliferation and resistance to apoptosis. Finally, delivery of synthetic miR-204 to the lungs of animals with PAH significantly reduced disease severity. This study uncovers a new regulatory pathway involving miR-204 that is critical to the etiology of PAH and indicates that reestablishing miR-204 expression should be explored as a potential new therapy for this disease.     

Differential signaling defects associated with the M201V polymorphism in the cysteinyl leukotriene type 2 receptor

The cysteinyl-leukotrienes (cysLTs) LTC(4), LTD(4), and LTE(4), are involved in a variety of inflammatory diseases, including asthma, and act on at least two distinct receptors, CysLT(1) and CysLT(2). Specific antagonists of CysLT(1) are currently used to control bronchoconstriction and inflammation in asthmatic patients. The potential role of CysLT(2) in asthma remains poorly understood. A polymorphism in the CysLT(2) gene, resulting in a single amino acid substitution (M201V), was found to be associated with asthma in three separate population studies. Here, we investigated whether the M201V mutation affected the affinity of CysLT(2) for its natural ligands and its signaling efficiency. Human embryonic kidney 293 cells were stably transfected with either wild-type (wt) or mutant (M201V) CysLT(2). Affinity of the M201V receptor for LTC(4) was reduced by 50%, whereas affinity for LTD(4) was essentially lost. LTC(4)-induced production of inositol phosphates (IPs) in M201V-expressing cells was significantly decreased at suboptimal concentrations of the ligand, but no difference was observed at high concentrations. In contrast, LTD(4)-induced IP production was 10- to 100-fold less in M201V- than in wt-expressing cells. Similar results were also observed with the transactivation of the interleukin-8 promoter induced by LTC(4) or LTD(4). Moreover, in contrast to wt-expressing cells, phosphorylation of nuclear factor κB p65 was absent in LTD(4)-stimulated M201V-expressing cells. Likewise, phosphorylation of c-Jun N-terminal kinase was not induced in LTD(4)-stimulated M201V cells, whereas activation of extracellular response kinase and p38 was maintained, at least at higher LTD(4) concentrations. Our results indicate that the M201V polymorphism drastically affects CysLT(2) responses to LTD(4) and less to LTC(4).     

Nornicotine application on cockroach dorsal unpaired median neurons induces two distinct ionic currents: implications of different nicotinic acetylcholine receptors

In order to increase our knowledge about the distribution of vitamins in the mammalian brain, we have developed a highly specific antiserum directed against retinoic acid with good affinity (10⁻⁸ M), as evaluated by ELISA tests. In the rat brain, no immunoreactive fibers containing retinoic acid were detected. Cell bodies containing retinoic acid were only found in the hypothalamus. This work reports the first visualization and the morphological characteristics of cell bodies containing retinoic acid in the mammalian paraventricular hypothalamic nucleus and in the dorsal perifornical region, using an indirect immunoperoxidase technique. The restricted distribution of retinoic acid in the rat brain suggests that this vitamin could be involved in very specific physiological mechanisms.     

Capacity utilization and the cost of primary care visits: Implications for the costs of scaling up health interventions

Objective

A great deal of international attention has been focussed recently on how much additional funding is required to scale up health interventions to meet global targets such as the Millennium Development Goals (MDGs). Most of the cost estimates that have been made in response have assumed that unit costs of delivering services will not change as coverage increases or as more and more interventions are delivered together. This is most unlikely. The main objective of this paper is to measure the impact of patient load on the cost per visit at primary health care facilities and the extent to which this would influence estimates of the costs and financial requirements to scale up interventions.

Methods

Multivariate regression analysis was used to explore the determinants of variability in unit costs using data for 44 countries with a total of 984 observations.

Findings

Controlling for other possible determinants, we find that the cost of an outpatient visit is very sensitive to the number of patients seen by providers each day at primary care facilities. Each 1% increase in patient through-put results, on average, in a 27% reduction in the cost per visit (p < 0.0001), which can lead to a difference of up to $30 in the observed costs of an outpatient visit at primary facilities in the same setting, other factors held constant.

Conclusion

Variability in capacity utilization, therefore, need to be taken into account in cost estimates, and the paper develops a method by which this can be done.     

OPG/membranous--RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation

The members of the OPG/RANK/RANKL (osteoprotegerin/receptor activator of nuclear factor kappaB/RANK ligand) triad are involved in various osteolytic pathologies such as bone tumors. Although many studies described the use of OPG during the treatment of bone diseases, its bioavailability and the mechanism by which the cells control the extracellular OPG remains blurred. The present work uses a strongly RANKL expressing cellular model to assess the becoming and the bioavailability of exogenous OPG in the context of its interactions with RANKL. The human kidney cell line 293, which initially expresses neither OPG nor RANKL, was stably transfected by the full length of mouse transmembranous form of RANKL (293RL). When OPG is incubated with 293RL cells, the extracellular concentration of OPG was strongly decreased in a time-dependent manner. The OPG disappearance was not inhibited by the addition of several proteases inhibitors, thus excluding any extracellular protease degradation. Contrary to previous results obtained on myeloma cells, which strongly express syndecan-1, the OPG disappearance was unaffected by the use of an antibody against syndecan-1. However, this event was abolished by an antibody against RANKL. These results, not necessarily conflicting, could be in relation with the expression level of the receptors in the two cellular models. In this context, an internalization process was put forward. Confocal microscopy demonstrated via the clathrin pathway an internalization of OPG mediated by RANKL. After being internalized, OPG was then degraded by the proteasome and the lysosome. A similar internalization phenomenon was also observed in osteoblast cells expressing physiologically RANKL, thus validating our data observed on 293RL cells. Western blotting analysis revealed that the half-life of RANKL was greatly reduced in the presence of OPG, pointing out that OPG binding to RANKL induces an enhancement of the ligand internalization. By the light of these results, the inhibitory effect of OPG on bone resorption can be explained not only by a decoy receptor function, competitor inhibitor of the RANK/RANKL binding, but also by the modulation of the RANKL half-life induced by OPG. Reciprocally, this modulation contributes to reduce the bioavailability of OPG.     

Physical Therapy (95)

Relationship between functional evaluation measures and self‐assessment in nonacute low back pain. (Concordia University, Montréal, Québec, Canada) Spine 2000;25:1817–1826. In this study, two hypotheses were examined: range of motion and velocity are controllable and inherently correlated with self‐assessment; complex spinal coordination patterns such as range of lordosis cannot be controlled and are independent of self‐assessment. Self‐assessment questionnaires were administered, and indexes of spinal motion and coordination were measured through skin marker kinematics. The correlation between self‐assessments and biomechanical measures was determined. Self‐assessments of function were significantly correlated with parameters prone to regulation: range of motion, velocity, and load lifted. In contrast, little correlation was found with measures of complex spinal coordination less susceptible to conscious or affective regulation, namely, range of lordosis, and estimated segmental mobility. This effect was magnified with increased load. Self‐assessment scores were significantly poorer among insurance referrals, regardless of functional status. Conclude that simple parameters of the functional examination, such as range of motion and velocity, are strongly correlated with cognitive state, and thus the information they supply is less than ideal. Complex spinal coordination is a better indicator of the degree of spinal dysfunction and enhances the process of differentiating between pain, disability, and functional impairment. Comment by Phillip S. Sizer Jr., MEd, PT. Numerous approaches to the evaluation of lumbar spine disorders have been proposed. Self‐reported disability scales are frequently implemented, but outcomes can be influenced by patient psychological factors. ROM tests have been frequently used for disability rating, but difficulty is encountered in detecting submaximal patient effort. These investigators evaluated the relationships between the Quebec Back Pain Disability Questionnaire and other self‐assessment measures, simple functional measures, and coordination patterns using the Spinoscope. From these measures the investigators attempted to elucidate a more accurate objective measure of biomechanical impairment independent from affective or self‐assessment influences. The outcomes illustrated that, while simple ROM and velocity measures were strongly correlated with subjective index scores, complex spinal coordination measures were not correlated with the same self‐assessment outcomes. Additionally, the authors suggested that simple ROM does not ensure biomechanical normalcy in movement. Furthermore, they suggested that complex coordination measures that include Range of Lordosis (ROL) and Estimate of Inter‐segmental Mobility (EISM) may serve as a better compliment to the clinical examination findings associated with lumbar conditions. Finally, they suggested that loading the spine during coordination testing further distances the outcomes from the subjective influence of pain and other factors. These measures may be superior to traditional measures for accurate diagnosis and return‐to‐work decisions with patients suffering from seemingly vague conditions, such as nonspecific low back pain.