Nicole Smith

TorsinA in PC12 cells: Localization in the endoplasmic reticulum and response to stress

Most cases of early‐onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the “ATPases associated with a variety of cellular activities” (AAA+) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor‐induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.

Magicin, a novel cytoskeletal protein associates with the NF2 tumor suppressor merlin and Grb2

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by bilateral vestibular schwannomas and meningiomas. Merlin, the neurofibromatosis 2 tumor suppressor protein, is related to the ERM (ezrin, radixin, moesin) proteins and, like its family members, is thought to play a role in plasma membrane–cytoskeletal interactions. We report a novel protein as a merlin-specific binding partner that we have named magicin (merlin and Grb2 interacting cytoskeletal protein) and show that the two proteins interact in vitro and in vivo as well as colocalize beneath the plasma membrane. Magicin is a 24 kDa protein that is expressed in many cell lines and tissues. Magicin, similar to merlin, associates with the actin cytoskeleton as determined by cofractionation, immunofluorescence and electron microscopy. Analysis of the magicin sequence reveals binding motifs for the adaptor protein Grb2. Employing affinity binding, blot overlay and co-immunoprecipitation assays, we demonstrate an interaction between Grb2 and magicin. In addition, merlin is capable of forming a ternary complex with magicin and Grb2. These results support a role for merlin in receptor-mediated signaling at the cell surface, and may have implications in the regulation of cytoskeletal reorganization.

The Coiled-coil Domain Is Required for HS1 to Bind to F-actin and Activate Arp2/3 Complex

HS1 (hematopoietic lineage cell-specific protein 1), a substrate of protein tyrosine kinases in lymphocytes, binds to F-actin, and promotes Arp2/3 complex-mediated actin polymerization. However, the mechanism for the interaction between HS1 and F-actin has not yet been fully characterized. HS1 contains 3.5 tandem repeats, a coiled-coil region, and an SH3 domain at the C terminus. Unlike cortactin, which is closely related to HS1 and requires absolutely the repeat domain for F-actin binding, an HS1 mutant with deletion of the repeat domain maintains a significant F-actin binding activity. On the other hand, deletion of the coiled-coil region abolished the ability of HS1 to bind to actin filaments and to activate the Arp2/3 complex for actin nucleation and actin branching. Furthermore, a peptide containing the coiled-coil sequence only was sufficient for F-actin binding. Within cells overexpressing green fluorescent protein-tagged HS1 proteins, wild type HS1 co-localizes with cortical F-actin at the cell leading edge, whereas mutants with deletion of either the coiled-coil region or the repeat domain diffuse in the cytoplasm. Immunoprecipitation analysis reveals that the coiled-coil deletion mutant binds poorly to F-actin, whereas the mutant without the repeat domain fails to bind to both Arp2/3 complex and F-actin. These data suggest that the HS1 coiled-coil region acts synergistically with the repeat domain in the modulation of the Arp2/3 complex-mediated actin polymerization.

Estimated fetal weight by ultrasound: a modifiable risk factor for cesarean delivery?

OBJECTIVE The purpose of this study was to investigate whether knowledge of ultrasound-obtained estimated fetal weight (US-EFW) is a risk factor for cesarean delivery (CD). STUDY DESIGN Retrospective cohort from a single center in 2009-2010 of singleton, term live births. CD rates were compared for women with and without US-EFW within 1 month of delivery and adjusted for potential confounders. RESULTS Of the 2329 women in our cohort, 50.2% had US-EFW within 1 month of delivery. CD was significantly more common for women with US-EFW (15.7% vs 10.2%; P < .001); after we controlled for confounders, US-EFW remained an independent risk factor for CD (odds ratio, 1.44; 95% confidence interval, 1.1-1.9). The risk increased when US-EFW was >3500 g (odds ratio, 1.8; 95% confidence interval, 1.3-2.7). CONCLUSION Knowledge of US-EFW, above and beyond the impact of fetal size itself, increases the risk of CD. Acquisition of US-EFW near term appears to be an independent and potentially modifiable risk factor for CD.

Determination of bisphenol-A levels in human amniotic fluid samples by liquid chromatography coupled with mass spectrometry

Bisphenol A (BPA) is one of the environmental endocrine-disrupting chemicals used widely in common consumer products. There is an increasing concern about human exposure to BPA, particularly in fetuses, due to the potential adverse effects related to the estrogenic activity of BPA. In assessing environmental exposure to BPA, it is essential to have a sensitive, accurate, and specific analytical method, particularly for low BPA levels in complex sample matrices. In this study, we developed and validated an accurate, sensitive, and robust liquid chromatography-mass spectrometry (LC-MS) method for determining the BPA concentrations in human amniotic fluid (AF). In this method, BPA and the internal standards (13)C(12) -BPA were extracted from 500 μL of human AF using solid-phase extraction. Calibration curves were linear over a concentration range of 0.3-100 ng/mL for BPA. The analytes were quantitatively determined using LC-MS operated in a negative electrospray ionization selected ion monitoring mode. This validated method has been used successfully in the clinical sample analysis of BPA in second-trimester AF specimens.

Protein:creatinine ratio in uncomplicated twin pregnancy.

OBJECTIVE Women with twin pregnancies may have higher rates of isolated proteinuria than do those with singletons. We compared protein-to-creatinine (P:C) ratios longitudinally through gestation in uncomplicated twin and singleton pregnancies. STUDY DESIGN P:C ratios were compared at 3 times points in 102 (51 twins, 51 singletons) healthy gravid patients who did not have preeclampsia develop, using linear and logistic regression techniques. RESULTS P:C ratio increased significantly over gestation in all patients. This increase was significantly greater in twins than in singletons. The odds of P:C ratio >0.19 was 3.5 times higher in twins between 34 and 38 weeks. CONCLUSION Women with uncomplicated twin pregnancies have greater protein excretion as measured by P:C ratios than do those with singletons. In early pregnancy, protein excretion is similar, but it diverges significantly by the latter third trimester. We suggest that normal values for proteinuria in twins may differ from those in singletons, and warrant further evaluation.

The E3 Ubiquitin Ligase Protein Associated with Myc (Pam) Regulates Mammalian/Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling in Vivo through N- and C-terminal Domains

Background: Pam and its homologs act as key regulators of axon guidance and outgrowth and synapse development. Results: Pam regulates mTORC1 signaling in vivo, and attenuated mTORC1 signaling may partly explain the axonal defects in Phr1-deficient mice. Conclusion: mTORC1-dependent and -independent mechanisms contribute to neurodevelopmental defects in Phr1 deficiency. Significance: Understanding how Pam regulates synapse development will have direct relevance for diverse aspects of neural development. Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1Δ8,9 and Phr1Magellan, with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1Δ8,9 and Phr1Magellan mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1Δ8,9 and Phr1Magellan mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1Δ8,9/Mag), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

Pregnancy-associated myocardial infarction: a report of two cases and review of the literature.

Background: Myocardial infarction in pregnancy carries high morbidity. Spontaneous coronary artery dissection is one etiology of infarction, and up to one third of cases may arise in the third trimester of pregnancy or within three months postpartum. Case: We report two cases of spontaneous coronary artery dissection, one at 34 weeks gestation and one postpartum. Both patients were diagnosed with angiography and treated medically and one required percutaneous coronary intervention, with good obstetric outcome and return of cardiac function. Conclusion: Myocardial infarction, and particularly spontaneous coronary artery dissection, should be in the differential diagnosis of pregnant women presenting with cardiac-type symptoms, despite perceived lack of risk factors. Angiography will aid in diagnosis, and multiple therapeutic modalities exist.

Identification of pathologically small fetuses using customized, ultrasound and population‐based growth norms

Fetal growth restriction is a strong risk factor for stillbirth. We compared the performance of three fetal growth curves – customized, ultrasound (Hadlock) and population – in identifying abnormally grown fetuses at risk of stillbirth.

Utilizing Longitudinal Measures of Fetal Growth to Create a Standard Method to Assess the Impacts of Maternal Disease and Environmental Exposure

Impaired or suboptimal fetal growth is associated with an increased risk of perinatal morbidity and mortality. By utilizing readily available clinical data on the relative size of the fetus at multiple points in pregnancy, including delivery, future epidemiological research can improve our understanding of the impacts of maternal, fetal, and environmental factors on fetal growth at different windows during pregnancy. This study presents mean and standard deviation ultrasound measurements from a clinically representative US population that can be utilized for creating Z-scores to this end. Between 2006 and 2012, 18, 904 non-anomalous pregnancies that received prenatal care, first and second trimester ultrasound evaluations, and ultimately delivered singleton newborns at Brigham and Women’s hospital in Boston were used to create the standard population. To illustrate the utility of this standard, we created Z-scores for ultrasound and delivery measurements for a cohort study population and examined associations with factors known to be associated with fetal growth. In addition to cross-sectional regression models, we created linear mixed models and generalized additive mixed models to illustrate how these scores can be utilized longitudinally and for the identification of windows of susceptibility. After adjustment for a priori confounders, maternal BMI was positively associated with increased fetal size beginning in the second trimester in cross-sectional models. Female infants and maternal smoking were associated with consistently reduced fetal size in the longitudinal models. Maternal age had a non-significant association with increased size in the first trimester that was attenuated as gestation progressed. As the growth measurements examined here are widely available in contemporary obstetrical practice, these data may be abstracted from medical records by investigators and standardized with the population means presented here. This will enable easy extension of clinical data to epidemiologic studies investigating novel maternal, fetal, and environmental factors that may impact fetal growth.