Prepregnancy body mass index and risk of childhood asthma.

BACKGROUND: Growing evidence suggests that maternal obesity may affect the intrauterine environment and increase a child's risk of developing asthma. We aim to investigate the relationship between prepregnancy obesity and childhood asthma risk. METHODS: Cohorts of children enrolled in Kaiser Permanente Northern California integrated healthcare system were followed from birth (2005-2014) to age 4 (n = 104,467), 6 (n = 63,084), or 8 (n = 31,006) using electronic medical records. Child's asthma was defined using ICD codes and asthma-related prescription medication dispensing. Risk ratios (RR) and 95% confidence intervals (95% CIs) for child's asthma were estimated using Poisson regression with robust error variance for (1) prepregnancy BMI categories (underweight [<18.5], normal [18.5-24.9], overweight [25-29.9], obese 1 [30-34.9], and obese 2/3 [≥35]) and (2) continuous prepregnancy BMI modeled using cubic splines with knots at BMI category boundaries. Models were adjusted for maternal age, education, race, asthma, allergies, smoking, gestational weight gain, child's birth year, parity, infant sex, gestational age, and child's BMI. RESULTS: Relative to normal BMI, RRs (95%CIs) for asthma at ages 4, 6, and 8 were 0.91 (0.75, 1.11), 0.95 (0.78, 1.16), and 0.97 (0.75, 1.27) for underweight, 1.06 (0.99, 1.14), 1.08 (1.01, 1.16), and 1.03 (0.94, 1.14) for overweight, 1.09 (1.00, 1.19), 1.12 (1.03, 1.23), 1.03 (0.91, 1.17) for obese 1, and 1.10 (0.99, 1.21), 1.13 (1.02, 1.25), 1.14 (0.99, 1.31) for obese 2/3. When continuous prepregnancy BMI was modeled with splines, child's asthma risk generally increased linearly with increasing prepregnancy BMI. CONCLUSIONS: Higher prepregnancy BMI is associated with modestly increased childhood asthma risk.

Maternal obesity and childhood asthma risk: Exploring mediating pathways.

BACKGROUND: Growing evidence for the effect of maternal obesity on childhood asthma motivates investigation of mediating pathways. OBJECTIVE: To investigate if childhood body mass index (BMI), gestational weight gain (GWG) and preterm birth mediate the association of maternal obesity on childhood asthma risk. METHODS: We used electronic medical records from mother-child pairs enrolled in Kaiser Permanente Northern California integrated healthcare system. Children were followed from their birth (2005-2014) until at least age 4 (n = 95,723), age 6 (n = 59,230) or age 8 (n = 25,261). Childhood asthma diagnosis at each age was determined using ICD-9/10 codes and medication dispensings. Prepregnancy BMI (underweight [<18.5], normal [18.5-24.9], overweight [25-29.9], obese [≥30] kg/m2 ) were defined using height and weight measurements close to the last menstrual period date. Child's BMI (Centers for Disease Control and Prevention BMI-for-age percentiles: underweight [<5th], normal [5th-85th], overweight [85th-95th], obese [>95th]) were obtained using anthropometric measurements taken the year preceding each follow-up age. GWG (delivery weight-prepregnancy weight) was categorised based on Institutes of Medicine recommendations (inadequate, adequate, excessive). Implementing first causal inference test (CIT) then causal mediator models (to decompose the natural direct and indirect effects), we examined the potential mediating effect of childhood BMI, GWG, and preterm birth on the association between prepregnancy BMI (continuous and categorical) and childhood asthma. RESULTS: Overall, risk of childhood asthma increased as prepregnancy BMI increased (age 4 risk ratio: 1.07, 95% confidence interval: 1.04, 1.09, per 5 kg/m2 increase in BMI; similar for age 6 and 8). CIT identified childhood BMI and preterm birth, but not GWG as potential mediators. Causal mediation models confirmed childhood BMI, but not preterm birth, as having a partial mediating effect. Results were similar for age 6 and 8, and when continuous mediators (instead of binary) were assessed. CONCLUSIONS: Childhood overweight/obesity has a modest mediating effect on the association between prepregnancy BMI and childhood asthma.

Matrix metalloproteinases 2 and 9 are elevated in human preterm laboring uterine myometrium and exacerbate uterine contractility†.

Matrix metalloproteinases 2 and 9 (MMP2/9) have previously been shown to be elevated in serum and amniotic fluid from women undergoing preterm birth. We performed experiments to determine the effects of MMP2/9 on uterine contraction and birth timing. Pregnant mice were injected daily with 50 mg/kg of SB-3CT or vehicle control beginning on gestational day 14-18 to determine if MMP2/9 inhibition would affect parturition timing. MMP2/9 expression in human myometrial tissue was determined by Simple Western (Wes) and semiquantitative western blot. Purified MMP2/9 and SB-3CT inhibitor were added to human myometrial strips to determine the effects of MMP2/9 on oxytocin-induced uterine contraction. Parturition was delayed in mice treated with MMP2/9 inhibitor SB-3CT. MMP2/9 protein levels were elevated in preterm laboring uterine myometrium. Gelatinase activity was confirmed in cell extracts and supernatants from immortalized and primary human uterine myometrial cells in culture. Addition of purified MMP2/9 increased the oxytocin-induced contractile response in myometrial tissue strips from pregnant women. In contrast, addition of the MMP2/9 inhibitor SB-3CT decreased the contractile response to oxytocin in a dose-dependent manner. These results suggest abnormal MMP2/9 expression affects the contractile state of the uterine myometrium to promote parturition and that MMP2/9 inhibition attenuates this effect.

Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization.

TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (<37 wk), leading to the hypothesis that these TREK-1 variants could interfere with TREK-1 function or expression. To investigate a potential role for these variants, immunofluorescence, cell surface assays, Western blots, and patch clamp were employed to study TREK-1 and TREK-1 variants expressed in HEK293T cells. The results of this study demonstrate that coexpression of TREK-1 with TREK-1 variants alters TREK-1 expression and suppresses channel function. Each variant affected TREK-1 in a disparate manner. In HEK293T cells coexpressing TREK-1 and each variant, TREK-1 membrane expression was diminished with compartmentalization inside the cell. When expressed alone, individual variants displayed channel properties that were significantly decreased compared to full-length TREK-1. In coexpression studies using patch clamp, basal TREK-1 currents were reduced by ∼64% (4.3 vs. 12.0 pA/pF) on average at 0 mV when coexpressed with each variant. TREK-1 currents that were activated by intracellular acidosis were reduced an average of ∼77% (21.4 vs. 94.5 pA/pF) at 0 mV when cells were transfected with TREK-1 and any one of the splice variants. These data correlate the presence of TREK-1 variants to reduced TREK-1 activity, suggesting a pathological role for TREK-1 variants in preterm labor.

Mechanical strain induced phospho-proteomic signaling in uterine smooth muscle cells.

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

LC/MS/MS data analysis of the human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor.

The data described in this article is the subject of an article in the American Journal of Physiology: Cell Physiology, titled "The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor, and Preterm Labor" (doi:10.1152/ajpcell.00198.2013) (Ulrich et al., 2013) [1]. The data described is a large scale mass spectrometry data set that defines the human uterine smooth muscle S-nitrosoproteome differences among laboring, non-laboring, preterm laboring tissue after treatment with S-nitrosoglutathione.

Blockade of extracellular NM23 or its endothelial target slows breast cancer growth and metastasis.

BACKGROUND: Nucleoside Diphosphate Kinase (NDPK), described as NM23 a metastasis suppressor, is found in the culture medium of cancer cells lines suggesting that the kinase may have an extracellular role. We propose that extracellular NM23 released from breast cancers in vivo stimulates tumor cell migration, proliferation and endothelial cell angiogenesis in support of metastasis development. METHODS: NM23 in the bloodstream of immunocompromised mice carrying human triple-negative breast cancers or in breast cancer patients was measured by ELISA. Primary and metastatic tumor development, the impact of blockade of NM23 and/or its stimulation of nucleotide receptors were measured using in vivo imaging. NM23 expression data in the Curtis breast dataset was examined to test our hypothesis that NM23 may play a mechanistic role in breast cancer development. RESULTS: SCID mice carrying metastatic MDA-MB-231Luc+ triple-negative human breast tumor cells elaborate NM23 into the circulation correlated with primary tumor growth. Treatment of mice with the NM23 inhibitor ellagic acid (EA) or the purinergic receptor antagonist MRS2179 slowed primary tumor growth. At 16 weeks following implantation, lung metastases were reduced in mice treated with EA, MRS2179 or the combination. Expression of NM23 in the Curtis breast dataset confirmed a likely role for NM23 in tumor metastasis. CONCLUSIONS: Extracellular NM23 may constitute both a biomarker and a therapeutic target in the management of breast cancer.

Integrin upregulation and localization to focal adhesion sites in pregnant human myometrium.

Focal adhesions are integrin-rich microdomains that structurally link the cytoskeleton to the extracellular matrix and transmit mechanical signals. In the pregnant uterus, increases in integrin expression and activation are thought to be critical for the formation of the mechanical syncytium required for labor. The aim of this study was to determine which integrins are upregulated and localized to focal adhesions in pregnant human myometrium. We used quantitative polymerase chain reaction, Western blotting, and confocal microscopy to determine the expression levels and colocalization with focal adhesion proteins. We observed increases in several integrin transcripts in pregnant myometrium. At the protein level, integrins such as α5-integrin (ITGA5), ITGA7, ITGAV, and ITGB3 were significantly increased during pregnancy. The integrins ITGA3, ITGA5, ITGA7, and ITGB1 colocalized with focal adhesion proteins in term human myometrium. These data suggest that integrins α3ß1, α5ß1, and α7ß1 are the most likely candidates to transmit mechanical signals from the extracellular matrix through focal adhesions in pregnant human myometrium.

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart.

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a K(D) for Ca(2+) of approximately 175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN(-)>I(-)>Cl(-), and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.

Complexin I is required for mammalian sperm acrosomal exocytosis.

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.

Molecular basis of mammalian gamete binding.

Despite the importance of fertilization for controlling human reproduction, regulating animal production, and promoting preservation of endangered species, the molecular basis underlying gamete binding and fertilization has been perplexing. More progress has been made in the mouse than in other mammals and, recently, targeted deletion of specific genes in the mouse has yielded intriguing results. This review will emphasize research performed by our laboratory and others done primarily with mouse gametes but will include some interesting observations from other mammals. Studies of murine fertilization indicate that oligosaccharides on the egg coat glycoprotein ZP3 bind sperm. The precise oligosaccharides that bind sperm are the subject of considerable debate. ZP3 also induces exocytosis of the sperm acrosome, allowing sperm to penetrate through the egg coat (zona pellucida). A number of candidate ZP3 receptors have been proposed and studies of beta1,4galactosyltransferase-I (GalT-I) are reviewed here in the most detail. Sperm from mice with a targeted deletion of GalT-I still are able to bind the zona pellucida but are unable to acrosome react and penetrate through the zona. Therefore, the unique role of GalT-I appears to be in signal transduction. GalT-I forms a complex with heterotrimeric G proteins and activates signaling, leading to exocytosis in sperm and in heterologous cells expressing GalT-I. Other signaling steps triggered by GalT-I are under active investigation; this receptor forms a complex with a protein kinase anchoring protein. After exocytosis of the acrosome, sperm penetrate the zona pellucida and fuse with the oocyte plasma membrane using ADAM family members on sperm and integrins on oocytes. These proteins, along with the tetraspanins on oocytes, may form a complex web at gamete fusion. Targeted deletion of specific genes in this putative complex has provided important information about their redundancy. After the oocyte is fertilized, the binding site for GalT-I is lost from ZP3, preventing additional sperm from binding to the zona pellucida. New technical advances and creative ideas offer the opportunity to make important advances and to solve the conundrum of fertilization.

CASK is in the mammalian sperm head and is processed during epididymal maturation.

Upon adhesion to the zona pellucida or egg extracellular matrix, sperm undergo regulated exocytosis of the acrosomal vesicle. CASK is an adaptor protein that has been implicated in coupling neuronal cell adhesion to regulated exocytosis. In neurons, this scaffolding molecule is associated with several types of transmembrane receptor complexes and connects cell adhesion molecules with ion channels, the actin cytoskeleton, and the cell's exocytotic machinery. We hypothesized CASK might also be an important link between zona pellucida binding and the sperm acrosome reaction. RT-PCR experiments indicated CASK is transcribed in mouse testis. The full size (120 kDa) CASK protein was present in testis from mouse and pig. Immunoblots of mature porcine and murine sperm revealed that the 120 kDa molecule was much less abundant than in testis but the antibody also recognized a group of smaller proteins migrating at 55-65 kDa. Immunofluorescence experiments indicated both the full length and smaller CASK immunoreactive products were found only in the acrosomal region of spermatids and mature sperm and not in other testicular cell types. CASK immunofluorescence was lost following the acrosome reaction. During epididymal maturation, the abundance of the full size CASK decreased and the CASK fragments increased. These results suggest that CASK may be proteolytically processed during epididymal maturation. Because sperm acquire the ability to bind the zona pellucida, acrosome react, and fertilize eggs during epididymal maturation, CASK processing may play a role in the acquisition of these functions.

A proteomic approach to identify phosphoproteins encoded by cDNA libraries.

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.