STK25 protein mediates TrkA and CCM2 protein-dependent death in pediatric tumor cells of neural origin.

The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin.

Functional consequences of necdin nucleocytoplasmic localization.

BACKGROUND: Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown. METHODOLOGY/PRINCIPAL FINDINGS: We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin ß1. Finally, exclusion of necdin from the nucleus triggered extensive cell death. CONCLUSIONS/SIGNIFICANCE: These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization.

Fine particulate matter (PM2.5) air pollution and immune status among women in the Seattle area.

Changes in immune status have been suggested as a possible biologic mechanism by which particulate matter (PM) air pollution could lead to adverse health effects. The authors studied associations between ambient PM2.5 and immune status among 115 postmenopausal, overweight women in the greater Seattle, Washington, area. The authors evaluated 3-day, 30-day, and 60-day average PM2.5 values in relation to inflammation markers (C-reactive protein, serum amyloid A, interleukin-6) and functional assays of cellular immunity (natural killer cell cytotoxicity, T-lymphocyte proliferation) at 3 time points for each woman during 1 year. Three-day averaged PM2.5 was inversely associated with anti-CD3-stimulated lymphocyte proliferation. There were no notable associations between the inflammation markers and PM2.5. If additional studies confirm our findings, then future health effect assessments for PM2.5 should consider changes in cellular immunity as an endpoint that may lead to overt clinical disease.

CCM2 mediates death signaling by the TrkA receptor tyrosine kinase.

The TrkA receptor tyrosine kinase is crucial for differentiation and survival of nerve-growth-factor-dependent neurons. Paradoxically, TrkA also induces cell death in pediatric tumor cells of neural origin, via an unknown mechanism. Here, we show that CCM2, a gene product associated with cerebral cavernous malformations, interacts with the juxtamembrane region of TrkA via its phosphotyrosine binding (PTB) domain and mediates TrkA-induced death in diverse cell types. Both the PTB and Karet domains of CCM2 are required for TrkA-dependent cell death, such that the PTB domain determines the specificity of the interaction, and the Karet domain links to death pathways. Downregulation of CCM2 in medulloblastoma or neuroblastoma cells attenuates TrkA-dependent death. Combined high expression levels of CCM2 and TrkA are correlated with long-term survival in a large cohort of human neuroblastoma patients. Thus, CCM2 is a key mediator of TrkA-dependent cell death in pediatric neuroblastic tumors.

Serum leptin concentrations and markers of immune function in overweight or obese postmenopausal women.

Experimental studies and case reports suggest a multifunctional role of leptin in immune function. However, clinical studies of leptin in healthy individuals with a comprehensive assessment of immunity are lacking. This study investigated associations between serum leptin concentrations and multiple biomarkers of cellular immunity and inflammation among 114 healthy postmenopausal, overweight, or obese women. Leptin was measured by RIA. C-reactive protein (CRP) and serum amyloid A (SAA) were measured by nephelometry. Flow cytometry was used to measure natural killer (NK) cell cytotoxicity and to enumerate and phenotype lymphocyte subsets. T-lymphocyte proliferation was assessed in response to phytohemagluttinin, as well as to anti-CD3 antibodies by the flow cytometric cell division tracking method. Multiple linear regression analysis with adjustment for confounding factors and log transformation, where appropriate, was used. Serum leptin concentrations were positively associated with serum CRP, SAA, and interleukin 6 (IL6) (P<0.0001, P=0.01, and P=0.04 respectively), more strongly among women with a body mass index (BMI) <30 kg/m(2). The associations were attenuated after adjustment for measured body composition, yet remained significant for CRP and SAA. No statistically significant associations were observed between leptin and NK cytotoxicity, lymphocyte subpopulations, or T-lymphocyte proliferation. This study fills an important gap in knowledge about the relationship between leptin concentrations and immune function in healthy individuals. Findings support an association between serum leptin and the inflammatory proteins CRP and SAA, which appears to be mediated only partly by adipose tissue. Our study does not support a link between leptin and other immune parameters among overweight or obese, but otherwise healthy postmenopausal women, perhaps because such effects are only present at low or deficient leptin concentrations.

Effect of exercise on in vitro immune function: a 12-month randomized, controlled trial among postmenopausal women.

Cross-sectional studies suggest that moderate physical activity is associated with enhanced resting immune function; however, few randomized controlled trials have investigated this link. We investigated the effect of 12-mo aerobic exercise, relative to stretching control, on in vitro immune function in a randomized, controlled trial of 115 postmenopausal, overweight, or obese sedentary women, aged 50-75 yr. The exercise goal was > or =45 min/day, 5 days/wk. Control women participated in 1 day/wk stretching classes. Immune markers (natural killer cell cytotoxicity, T-lymphocyte proliferation, immune cell counts and phenotypes, and serum immunoglobulins) were assessed at baseline, 3 mo, and 12 mo under strict blood-draw criteria. General estimation equations evaluated intervention effects at 3 and 12 mo, controlling for baseline. Of the 115 women who began the trial, blood samples were available from 109 at 3 mo (95%) and 108 at 12 mo (94%). From baseline to 12 mo, the exercise group participated in 87% of the prescribed physical activity minutes per week and increased maximal O(2) uptake by 13.8%; controls experienced no change in fitness. The main outcomes, natural killer cell cytotoxicity and T-lymphocyte proliferation, did not differ between groups at 3 and 12 mo. Secondary outcome and subgroup (e.g., stratification by baseline categories of body mass index, immune status, C-reactive protein, and age) analyses did not show any clear patterns of association. This 12-mo randomized, controlled trial showed no effect of aerobic exercise on in vitro immune function, despite excellent retention, high adherence, and demonstrable efficacy of the exercise intervention.

Associations between healthy eating patterns and immune function or inflammation in overweight or obese postmenopausal women.

BACKGROUND: The link between poor nutritional status and impaired immune function is well established; however, most studies have focused on individual nutrients instead of overall dietary patterns. OBJECTIVE: Our objective was to investigate associations between 3 indexes of overall diet quality [the Diet Quality Index (DQI), the DQI including supplementary calcium (DQI-Ca), and the Healthy Eating Index (HEI)] and biomarkers of inflammation and immunity. DESIGN: This cross-sectional study included 110 overweight or obese postmenopausal women. Dietary intake measured by food-frequency questionnaire was used to calculate diet quality scores. C-reactive protein (CRP) and serum amyloid A (SAA) were measured by latex-enhanced nephelometry. Flow cytometry was used to measure natural killer (NK) cell cytotoxicity and to enumerate and phenotype lymphocyte subsets. T lymphocyte proliferation was assessed by (3)H-thymidine incorporation as well as by the carboxyfluorescein-succinimidyl ester method of cell division tracking. Multivariable-adjusted linear regression analysis was used to investigate associations between diet quality scores and markers of inflammation and immune function. RESULTS: Higher diet quality was associated with increased proportions of cytotoxic and decreased proportions of helper T lymphocytes. CRP and SAA concentrations were higher among women with a lower-quality diet; these associations became nonsignificant after adjustment for body mass index or percentage body fat. We observed limited evidence for an association between healthy eating patterns and greater lymphocyte proliferation and no evidence for an association with NK cell cytotoxicity. CONCLUSION: Our results provide limited evidence that healthy eating patterns contribute to enhanced immune function and reduced inflammation in overweight and obese postmenopausal women.

Factors affecting urinary myoglobin stability in vitro.

Urine myoglobin concentrations are measured clinically to assess rhabdomyolysis and the related risk of renal damage. We studied urine myoglobin concentrations in vitro to explore the factors affecting stability. Myoglobin was very unstable in urine specimens, especially below pH 6.5, and its immunoreactivity deteriorated rapidly with increasing temperatures. The deterioration rate was influenced greatly by urine myoglobin concentration, suggesting rate-limiting kinetics. Myoglobin in acidic phosphate-buffered saline was significantly more stable than in acidic urine, indicating that urinary factors in addition to pH are involved in myoglobin instability. These unidentified urinary factors had a molecular weight of less than 10 kd. Our results provide additional insight into the mechanism involved in the instability of the urine myoglobin concentration. Understanding the stability of myoglobin in the preanalytic in vitro phase and its potential in vivo instability is essential in assuring the reliability and clinical usefulness of urine myoglobin measurements.

A genome wide screening approach for membrane-targeted proteins.

Membrane-associated proteins are critical for intra- and intercellular communication. Accordingly approaches are needed for rapid and comprehensive identification of all membrane-targeted gene products in a given cell or tissue. Here we describe a modification of the yeast Ras recruitment system to this end and designate the modified approach the Ras membrane trap (RMT). A pilot RMT screen was carried out on the central nervous system of the mollusk Lymnaea stagnalis, a model organism from a phylum that still lacks a representative with a sequenced genome. 112 gene products were identified in the screen of which 79 lack assignable homologs in available data bases. Currently available annotation tools predicted membrane association of only 45% of the 112 proteins, although experimental verification in mammalian cells confirmed membrane association for all clones tested. Thus, genome annotation using currently available tools is likely to underpredict representation of membrane-associated gene products. The 32 proteins with known homologies include many targeted to the endoplasmic reticulum or the nucleus, thus RMT provides a tool that can cover intracellular membrane proteomes. Two sequences were found to represent gene families not found to date in invertebrate genomes, emphasizing the need for whole genome sequences from mollusks and indeed from representatives of all major invertebrate phyla.

Neurotrophic activities of trk receptors conserved over 600 million years of evolution.

The trk family of receptor tyrosine kinases is crucial for neuronal survival in the vertebrate nervous system, however both C. elegans and Drosophila lack genes encoding trks or their ligands. The only invertebrate representative of this gene family identified to date is Ltrk from the mollusk Lymnaea. Did trophic functions of trk receptors originate early in evolution, or were they an innovation of the vertebrates? Here we show that the Ltrk gene conserves a similar exon/intron order as mammalian trk genes in the region encoding defined extracellular motifs, including one exon encoding a putative variant immunoglobulin-like domain. Chimeric receptors containing the intracellular and transmembrane domains of Ltrk undergo ligand-induced autophosphorylation followed by MAP kinase activation in transfected cells. The chimeras are internalized similarly to TrkA in PC12 cells, and their stimulation leads to differentiation and neurite extension. Knock-down of endogenous Ltrk expression compromises outgrowth and survival of Lymnaea neurons cultured in CNS-conditioned medium. Thus, Ltrk is required for neuronal survival, suggesting that trophic activities of the trk receptor family originated before the divergence of molluscan and vertebrate lineages approximately 600 million years ago.

Quick parathormone assay in the surgical management of hyperparathyroidism.

BACKGROUND: Among the various new technologies in the field of parathyroid surgery is intraoperative quick parathormone measurements. OBJECTIVES: To evaluate the contribution of QPTH measurements during parathyroidectomy to the achievement of higher success rates. METHODS: QPTH assay using Immulite Turbo Intact PTH was measured in 32 patients undergoing parathyroidectomy: 30 for primary and 2 for secondary hyperparathyroidism. QPTH levels were measured at time 0 minutes (before incision) and at 10, 20, and 30 minutes after excision of the hyperfunctioning gland. Only a drop of 60% or more from the 0' level was considered to be a positive result. RESULTS: The mean QPTH level at time 0' for PHPT patients was 38.12 +/- 25.15 pmol/L (range 9.1-118 pmol/L). At 10 minutes post-excision of the hyperfunctioning gland (or glands), QPTH dropped by a mean of 73.80% to 9.89 +/- 18.78 pmol/L. CONCLUSIONS: Intraoperative QPTH level measurement is helpful in parathyroid surgery. A drop of 60% or more from 0' level indicates a successful procedure, and further exploration should be avoided.

Cyclic changes of plasma spermine concentrations in women.

Based on previous studies which suggest that blood polyamines fluctuate during the menstrual cycle, the present study was set to determine whether plasma concentrations of the polyamine spermine show menstrual cycle-associated changes and if so, how these changes relate to phasic variations in other female hormones. Blood samples were collected from a group of 9 healthy women of various ages at 5 defined periods during their menstrual cycle including 1 woman on oral contraceptives. Spermine concentrations were determined in plasma acid extracts by reversed-phase high performance liquid chromatography method. Plasma estradiol, LH and FSH were measured by microparticle enzyme immunoassay using an automatic analyzer. Spermine concentrations, 104.4 +/- 12.2 nmol/ml at 1-3 day of the cycle, were increased transiently with a peak (263.8 +/- 22.1 nmol/ml) at 8-10 day and declined to 85.4 +/- 29.8 nmol/ml by 21-23 day of the cycle. The peak spermine concentrations coincided with the first increase in plasma estrogen levels. The individual variations in the temporal profile of spermine concentrations were of similar magnitude as individual differences in other female hormones. We conclude that: a) Plasma spermine concentrations undergo distinct cyclic alterations during the menstrual cycle with peak concentrations coinciding with the first estradiol increase, and b) Peak plasma spermine concentrations occur during the follicular phase, just prior to ovulation, during the period of rapid endometrial growth.

The p75 neurotrophin receptor interacts with multiple MAGE proteins.

The p75 neurotrophin receptor has been implicated in diverse aspects of neurotrophin signaling, but the mechanisms by which its effects are mediated are not well understood. Here we identify two MAGE proteins, necdin and MAGE-H1, as interactors for the intracellular domain of p75 and show that the interaction is enhanced by ligand stimulation. PC12 cells transfected with necdin or MAGE-H1 exhibit accelerated differentiation in response to nerve growth factor. Expression of these two MAGE proteins is predominantly cytoplasmic in PC12 cells, and necdin was found to be capable of homodimerization, suggesting that it may act as a cytoplasmic adaptor to recruit a signaling complex to p75. These findings indicate that diverse MAGE family members can interact with the p75 receptor and highlight type II MAGE proteins as a potential family of interactors for signaling proteins containing type II death domains.