Maternal periconceptional exposure to drinking water disinfection by-products and neural tube defects in offspring.

BACKGROUND: Associations between maternal periconceptional exposure to disinfection by-products (DBPs) in drinking water and neural tube defects (NTDs) in offspring are inconclusive, limited in part by exposure misclassification. METHODS: Maternal interview reports of drinking water sources and consumption from the National Birth Defects Prevention Study were linked with DBP concentrations in public water system monitoring data for case children with an NTD and control children delivered during 2000-2005. DBPs analyzed were total trihalomethanes, the five most common haloacetic acids combined, and individual species. Associations were estimated for all NTDs combined and selected subtypes (spina bifida, anencephaly) with maternal periconceptional exposure to DBPs in public water systems and with average daily periconceptional ingestion of DBPs accounting for individual-level consumption and filtration information. Mixed effects logistic regression models with maternal race/ethnicity and educational attainment at delivery as fixed effects and study site as a random intercept were applied. RESULTS: Overall, 111 case and 649 control children were eligible for analyses. Adjusted odds ratios for maternal exposure to DBPs in public water systems ranged from 0.8-1.5 for all NTDs combined, 0.6-2.0 for spina bifida, and 0.7-1.9 for anencephaly; respective ranges for average daily maternal ingestion of DBPs were 0.7-1.1, 0.5-1.5, and 0.6-1.8. Several positive estimates (≥1.2) were observed, but all confidence intervals included the null. CONCLUSIONS: Using community- and individual-level data from a large, US, population-based, case-control study, we observed statistically nonsignificant associations between maternal periconceptional exposure to total and individual DBP species in drinking water and NTDs and subtypes.

Conservation of the 3'-untranslated regions of calmodulin mRNAs in cetaceans.

Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5'- and 3'-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3'- untranslated region (3'UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3'UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3'UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3'UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3'UTR sequences. Such high conservation of the 3'UTRs suggests a specialized and significant function for this region in mammals.

Sequence homology of the 3'-untranslated region of calmodulin III in mammals.

Calmodulin (CaM) has three separate nonallelic genes that encode for three identical proteins. These genes differ considerably in the 5'- and 3'-untranslated regions (UTR) and in the promoter region. The sequence of the 3'-UTR of CaM III gene for rat and mouse was completed and compared to the human sequence. The rat and mouse 3'-UTR region had an identity of approximately 80% with the human. Three common polyadenylation signals in the 3'-UTR may account for the multiple CaM III transcripts. Although the untranslated regions are distinctively different for the three CaM genes, these regions are highly conserved among mammals. This high sequence conservation suggests an important function in the localization or regulation of CaM mRNA.

Evolutionary aspects of calmodulin.

Calmodulin (CaM) is a major cellular sensor of calcium signaling, interacts with numerous proteins associated with cellular second messenger systems (e.g., cyclic AMP, nitric oxide), and is associated with neurosecretory activity. An identical CaM protein consisting of four helix-loop-helix regions that arose by gene duplication is encoded by three nonallelic mammalian genes that are some of the most highly conserved genes known. Differential tissue and cellular expression of each CaM suggest unique functions that promote strong selective preservation of these replicate, yet distinct, CaM genes in mammals. Each gene displays the same exon-intron arrangement but is characterized by distinct promoter elements and by unique 5'- and 3'-untranslated regions that are highly conserved among human, rat, and mouse. These distinct untranslated regions may permit regulation of CaM levels at discrete cellular sites during differentiation and in highly specialized cell types such as neurons.

Calculation and verification of the ages of retroprocessed pseudogenes.

To verify the existence of processed pseudogenes in different primates and their correlation with the estimated age of divergence, selected regions of processed pseudogenes of alpha-enolase, calmodulin II (CALMII), and argininosuccinate synthetase (AS) were amplified by the polymerase chain reaction (PCR) using DNA of blood samples. Published primate divergence times from the accepted paleontological records and the age of the pseudogenes based on molecular clock calculations were compared to data obtained by detection of PCR products exhibiting the expected amplicon size of the pseudogene region. For the alpha-enolase and the CALMII pseudogenes Psi(2), and Psi(3), calculated divergence times were 11, 19, and 36 Myr, respectively. For the AS pseudogenes Psi(1), Psi(3), and Psi(7), the divergence times were calculated to be 21, 25, and 16 Myr, respectively. Primer design and the annealing temperature are critical factors in the detection of pseudogenes in different species and impact greatly on the interpretation of the PCR analysis. The estimated divergence times of the selected pseudogenes utilizing calculations based on the molecular clock theory correlated well with experimental PCR detection of the selected pseudogenes represented in this study.

The human PCPH proto-oncogene: cDNA identification, primary structure, chromosomal mapping, and expression in normal and tumor cells.

We identified a human cDNA encoding a 47-kDa protein that shares 78% and 87% identity with the products of the Syrian hamster and mouse PCPH proto-oncogenes respectively. The human homolog was localized by radiation-hybrid mapping to chromosome band 14q24.3, a region syntenic to the Pcph location on mouse chromosome 12. Northern analyses revealed that PCPH mRNA was widely distributed in normal human adult tissues, but its expression varied significantly among human tumor cells and cell lines of several tissue types, regardless of the level of expression in the corresponding normal tissues. The highest levels of PCPH mRNA and protein were detected in kidney and liver. However, PCPH was not expressed in the majority of human neoplasms tested, including kidney tumors. These data provide suggestive evidence for a possible association of the lack of PCPH expression to the neoplastic phenotype of human tumor cells. Our results should prove instrumental in designing studies to define the cellular function of the human PCPH proto-oncogene.

Radiation hybrid mapping of genes in the lithium-sensitive wnt signaling pathway.

Lithium, an effective drug in the treatment of bipolar disorder, has been proposed to disrupt the Wnt signaling pathway. To facilitate analysis of the possible involvement of elements of the Wnt pathway in human bipolar disorder, a high resolution radiation hybrid mapping (RHM) of these genes was performed. A fine physical location has been obtained for Wnt 7A, frizzled 3, 4 and 5, dishevelled 1, 2 and 3, GSK3beta, axin, alpha-catenin, the Armadillo repeat-containing genes (delta-catenin and ARVCF), and a frizzled-like protein (frpHE) using the Stanford Human Genome Center (SHGC) G3 panel. Most of these genes were previously mapped by fluorescence in situ hybridization (FISH). Frizzled 4, axin and frpHE did not have a previous chromosomal assignment and were linked by RHM to chromosome markers, SHGC-35131 at 11q22.1, NIB1488 at 16p13.3 and D7S2919 at 7p15.2, respectively. Interestingly, some of these genes were found to map within potential regions underlying susceptibility to bipolar disorder and schizophrenia as well as disorders of neurodevelopmental origin. This alternative approach of establishing the precise location of selected genetic components of a candidate pathway and determining if they map within previously defined susceptibility loci should help to identify plausible candidate genes that warrant further analysis through association and mutational scanning.

Sequence motifs for calmodulin recognition.

Calmodulin (CaM) is recognized as a major calcium sensor and orchestrator of regulatory events through its interaction with a diverse group of cellular proteins. Many investigations have focused on defining the region of interaction between CaM and its cellular targets and the action of CaM on target protein function. Because CaM can bind with high affinity to a relatively small alpha-helical region of many proteins, success in clearly defining the essential elements of CaM binding motifs seems feasible and should provide a means of identifying CaM binding proteins. Three recognition motifs for CaM interaction are discussed in the context of experimental investigations of a variety of CaM target proteins. A modified version of the IQ motif as a consensus for Ca2+-independent binding and two related motifs for Ca2+-dependent binding, termed 18-14 and 1-5-10 based on the position of conserved hydrophobic residues, are proposed. Although considerable sequence diversity is observed among the different binding regions, these three classes of recognition motifs exist for many of the known CaM binding proteins.

Biological response modifiers and chemotherapeutic agents that alter interleukin 2 activities.

IL2 immunotherapy alone or with LAK cells represents a novel approach to the treatment of metastatic cancers. Similarly, other BRMs and classical chemotherapeutic drugs through their molecular effects on the components of the immune system reveal new and exciting prospects for the better use of these agents. Both approaches converge in that they restore balance among numerous components of the immune surveillance system. This review raises the possibility of improved protocols through the judicious use of these agents and stresses the need for further investigation of combined use of chemotherapy and immunotherapy.

ATP-induced secretion in PC12 cells and photoaffinity labeling of receptors.

Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2-type purinergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATP gamma S, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4- was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate. The stimulatory effects of 12-O-tetradecanoylphorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [alpha-32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP gamma S and BzATP but not GTP blocked labeling of the proteins by [32P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P2 receptor responsible for ATP-stimulated secretion in these cells.

Small membrane-associated GTP-binding proteins of catecholamine-secreting cells.

1. Four GTP-binding proteins (23-27 kDa) were identified in membranes from PC12 cells by [alpha 32P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels. 2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K(+)-depolarization or even after addition of Ca2+ to digitonin-permeabilized cells. 3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca2+. 4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity. 5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane. 6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

Hydrolysis-resistant GTP analogs stimulate catecholamine release from digitonin-permeabilized PC12 cells.

The effect of the hydrolysis-resistant GTP analogs, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanylyl imidodiphosphate (GMPPNP), on norepinephrine (NE) secretion from digitonin-permeabilized rat pheochromocytoma cells, PC12, was examined. Although secretion in the presence of saturating Ca2+ (10 microM) was not affected by GTP gamma S or GMPPNP, secretion in the absence of Ca2+ was stimulated by these GTP analogs. Secretion induced by saturating concentrations of GTP gamma S or GMPPNP was approximately 80% of that induced by 10 microM Ca2+. Half-maximum stimulation was induced by 30 microM GTP gamma S or GMPPNP. Both Ca2(+)-stimulated and GTP gamma S-stimulated secretion were ATP dependent and inhibited by N-ethylmaleimide. The GTP gamma S-stimulated secretion of NE from permeabilized PC12 cells does not appear to result from either the release of Ca2+ or the activation of protein kinase C. Activation of protein kinase C by pretreatment of intact cells with 12-O-tetradecanoylphorbol 13-acetate caused a 50% increase in both Ca2(+)-stimulated and GTP gamma S-stimulated secretion. Cholera and pertussis toxins did not affect Ca2(+)-stimulated or GTP gamma S-stimulated NE secretion. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and GTP inhibited GTP gamma S-stimulated secretion but not Ca2(+)-stimulated secretion. The inability of GDP beta S to inhibit Ca2(+)-stimulated secretion indicates that the process affected by GTP gamma S is not an essential step in the Ca2(+)-stimulated pathway.

Involvement of arginine residues in the activation of calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase.

Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC 3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic AMP and cyclic GMP with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-1 min-1. Other arginine-modifying reagents, 1,2-cyclohexanedione and 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 microM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of CaM during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of CaM necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-14C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of CaM stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and CaM and that controlled modification can selectively alter CaM-stimulated enzyme activity.

Interaction of calmodulin with the particulate fraction of cardiac muscle.

Tritiated calmodulin (T-CM) was bound to the EGTA-treated particulate fraction of cardiac muscle in a calcium-dependent manner with half-maximal binding occurring between 0.8 to 1.2 microM calcium. Binding exhibited high specificity at an optimum pH of 7.4-7.6. An excess of parvalbumin and other globular proteins did not displace T-CM. The Kd for the interaction was 2.5 +/- 0.83 microM. Binding was trypsin-sensitive, inhibited by high ionic strength and was heat inactivated at a midpoint of 48 - 50 degrees C. Competitive displacement of T-CM occurred with unlabeled troponin C and calmodulin over the same concentration range. The first-order rate constant of T-CM dissociation was 3.27 min-1. Calcium-dependent binding of T-CM was inhibited equally by both mepacrine and trifluoperazine with 50 percent inhibition occurring at 70 microM.

Comparative studies of computerized tomography and mediastinoscopy for the staging of bronchogenic carcinoma.

The accuracy of mediastinal computerized tomographic scans for the staging of bronchogenic carcinoma varies between institutions. In the present study, the sensitivity rate was 57 percent, the specificity rate 69 percent, and the overall accuracy rate 64 percent, all of which were generally lower than rates reported in the recent literature. Different scanning equipment, diagnostic criteria, and patient populations may all contribute to this variance. The data in this report suggest that tumor histologic type and location also influenced the accuracy of computerized tomography. On the basis of this study and review of the literature, it is recommended that any given institution assess the accuracy of its own computerized tomographic mediastinal scans before substituting scanning for mediastinoscopy in the preoperative staging of bronchogenic carcinoma.

Characterization of calcium-dependent membrane binding proteins of brain cortex.

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr-32 000 and -34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

Particulate cyclic 3',5'-nucleotide phosphodiesterase and calmodulin of cardiac muscle.

Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl2.