Direct, indirect and pleiotropic effects of candidate genes on internalizing disorder psychopathology.

BACKGROUND: Twin studies of internalizing disorders suggest that their high co-morbidity is partially explained by shared genetic risk. Few studies have investigated pleiotropic effects of well-validated candidate genes across phenotypes. METHOD: Subjects were 928 Caucasian patients who presented to an out-patient clinic specializing in the assessment and treatment of anxiety and mood disorders. We constructed latent dimensional phenotypes across the internalizing spectrum (neuroticism, extraversion, depression, generalized anxiety, panic/agoraphobia, social phobia, post-traumatic stress, and obsessions-compulsions) by combining diagnostic criteria with other clinical indicators. We selected multiple variants in four evidence-based candidate genes (SLC6A4, COMT, GAD1, RGS2) with previously reported effects on several of these phenotypes. We conducted genetic association testing of their direct and indirect effects as well as gene × stress interactions (G × E). RESULTS: We detected 19 nominally significant main effect associations for the 10 polymorphisms tested among the eight phenotypes (24%). These were generally phenotype non-specific, showing pleiotropic effects across multiple domains. The majority of observed sharing was between depression, panic disorder, and post-traumatic stress disorder. Some of these were best explained by mediational models in which genes increase liability for disorders indirectly via their effects on temperament. Limited G × E effects were detected between variants in SLC6A4 and both panic/agoraphobia and post-traumatic stress. CONCLUSIONS: Examining just a few candidate genes for their potential roles in internalizing phenotypes, we found moderate support for the shared effects of several polymorphisms. These findings highlight the richness and complexity by which genes potentially contribute to psychopathology via pleiotropy, moderation by stress, and mediation by temperament.

Operation of the "Small" BioAMS Spectrometers at CAMS: Past and Future Prospects.

A summary of results from the solid samples run on our compact 1 MV AMS system over its 13.5 years of operation is presented. On average 7065 samples per year were measured with that average dropping to 3278 samples per year following the deployment of our liquid sample capability. Although the dynamic range of our spectrometer is 4.5 orders in magnitude, most of the measured graphitic samples had 14C/C concentrations between 0.1 and 1 modern. The measurements of our ANU sucrose standard followed a Gaussian distribution with an average of 1.5082 ± 0.0134 modern. The LLNL biomedical AMS program supported many different types of experiments, however, the large majority of samples measured were derived from animal model systems. We have transitioned all of our biomedical AMS measurements to the recently installed 250 kV SSAMS instrument with good agreement compared in measured 14C/C isotopic ratios between sample splits. Finally, we present results from replacement of argon stripping gas with helium in the SSAMS with a 22% improvement in ion transmission through the accelerator and high-energy analyzing magnet.

A longitudinal examination of psychosocial impairment across the anxiety disorders.

BACKGROUND: Anxiety disorders are highly prevalent disorders associated with substantial psychosocial impairment, but few studies have examined impairment within specific anxiety disorders. Furthermore, it is unclear how change in different types of anxiety has an impact on change in impairment, particularly given high rates of co-morbidity. The current study assessed the temporal associations of impairment and symptoms of three common anxiety disorders in a large, diagnostically heterogeneous clinical sample. METHOD: Data were collected from 606 treatment-seeking individuals at an anxiety clinic, most of whom subsequently enrolled in cognitive-behavioral therapy. Symptoms of panic, social anxiety and generalized anxiety disorder (GAD), as well as levels of impairment, were assessed three times over 2 years. In addition to examining levels of impairment across diagnostic groups, latent growth modeling was used to evaluate the longitudinal associations of anxiety symptoms and impairment. RESULTS: Those with a principal diagnosis of GAD reported higher levels of impairment in some domains at baseline; however, at follow-up assessments individuals with social anxiety disorder reported greater impairment than those with panic disorder. Anxiety symptoms and impairment both declined over time. Change in all three anxiety symptoms was closely associated with change in impairment, but only GAD remained a significant (positive) predictor of change in impairment after accounting for co-morbidity. CONCLUSIONS: Impairment and all three anxiety disorders were closely associated, both cross-sectionally and longitudinally. Because change in GAD was most specifically related to change in impairment, treatment for those with multiple anxiety disorders could focus on treating GAD symptoms first or treating transdiagnostic processes.

Role of serum IgA. Hepatobiliary transport of circulating antigen.

The IgA mediated hepatobiliary excretion of antigen from the circulation was studied using a radiolabeled haptenated protein (dinitrophenyl-human serum albumin) injected intravenously in mice together with monoclonal anti-dinitrophenyl antibodies of different immunoglobulin classes. Antibodies were obtained from ascitic fluids of mice bearing the MOPC315 myeloma (IgA), or immune spleen cell hybridomas (IgG and IgM). IgA antibody brought about the transport of large amounts of antigen from the circulation to the bile during 1-3h. Analysis of bile by gel filtration showed that a large part of the transported antigen remained intact and complexed with IgA. Neither IgA of different specificity nor anti-dinitrophenyl IgM medicated biliary transport of antigen. With anti-dinitrophenyl IgG, only small amounts of low molecular weight fragments of labeled antigen were found in he bile. Preformed immune complex of radiolabeled antigen and IgA antibody were rapidly transported from the circulation to the bile, resulting in threefold-higher levels of radioactivity in bile than in serum. It is proposed that an important function of serum IgA is to mediate the hepatobiliary excretion of corresponding circulating antigens.

Direct measurements of ²²Na(p,γ)²³Mg resonances and consequences for ²²Na production in classical novae.

The radionuclide 22Na is a potential astronomical observable that is expected to be produced in classical novae in quantities that depend on the thermonuclear rate of the 22Na(p,γ)23Mg reaction. We have measured the strengths of low-energy 22Na(p,γ)23Mg resonances directly and absolutely using a radioactive 22Na target. We find the strengths of resonances at Ep=213, 288, 454, and 610 keV to be higher than previous measurements by factors of 2.4-3.2, and we exclude important contributions to the rate from proposed resonances at Ep=198, 209, and 232 keV. The 22Na abundances expected in the ejecta of classical novae are reduced by a factor of ≈2.

Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons.

We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor III (ECMR III), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HT1080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonoverlapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O- and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.

Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex.

Analysis of the heteromeric DNA binding protein GABP has revealed the interaction of two distinct peptide sequence motifs normally associated with proteins located in different cellular compartments. The alpha subunit of GABP contains an 85-amino acid segment related to the Ets family of DNA binding proteins. The ETS domain of GABP alpha facilitates weak binding to DNA and, together with an adjacent segment of 37 amino acids, mediates stable interaction with GABP beta. The beta subunit of GABP contains four imperfect repeats of a sequence present in several transmembrane proteins including the product of the Notch gene of Drosophila melanogaster. These amino-terminal repeats of GABP beta mediate stable interaction with GABP alpha and, when complexed with GABP alpha, directly contact DNA. These observations provide evidence for a distinct biochemical role for the 33-amino acid repeats, and suggest that they may serve as a module for the generation of specific dimerization interfaces.

Surface-specific effects of a PPARgamma agonist, darglitazone, on bone in mice.

It has been hypothesized that activation of peroxisome-proliferator-activated receptor-gamma (PPARgamma) by thiazolidinedione drugs can increase adipogenesis at the expense of osteogenesis, leading to bone loss. However, the reported skeletal effects of these compounds are varied and their effects on cortical bone are unknown. In this study, we examined the changes in both cancellous and cortical bone of 6-month-old male mice treated with darglitazone, a potent and selective PPARgamma agonist, at 10 mg/kg/day by dosing the compound in a food mixture for 2 or 8 weeks. At 2 weeks, we observed significantly increased marrow adipose tissue area, decreased trabecular bone density of distal femur, and decreased surface referent bone formation rate of lumbar vertebrae in the mice treated with darglitazone compared with controls. At 8 weeks, lower cancellous bone mass was seen at both distal femurs and lumbar vertebrae of the mice treated with darglitazone. In addition, mineralizing surface was significantly lower, whereas osteoclast surface and number were significantly higher in the lumbar vertebrae of darglitazone-treated mice. At the femoral diaphysis, darglitazone treatment caused bone loss on the endocortical surface. Interestingly, periosteal mineral apposition rate and surface referent bone formation rate were significantly increased in darglitazone-treated mice. In bone marrow cell cultures, darglitazone suppressed alkaline phosphatase activity, osteoblastic gene expression, and mineralized nodule formation while increasing adipogenic gene expression and lipid accumulation. In summary, darglitazone enhanced adipogenesis and caused cancellous bone loss by increasing bone resorption and decreasing bone formation in mice. In addition, darglitazone induced cortical bone loss on the endocortical surface but increased bone formation on the periosteal surface. These data suggest that PPARgamma plays a role in regulating bone resorption and formation and reveal surface-specific effects of a PPARgamma agonist on bone.

Production and separation of carrier-free 7Be.

A high-purity carrier-free (7)Be was efficiently isolated following proton bombardment of a lithium hydroxide-aluminum target. The separation of beryllium from lithium and aluminum was achieved through a hydrochloric acid elution system utilizing cation exchange chromatography. The beryllium recovery, +99%, was assessed through gamma spectroscopy while the chemical purity was established by mass spectrometry. The decontamination factors of beryllium from lithium and aluminum were determined to be 6900 and 300, respectively.

Source identification and distribution reveals the potential of the geochemical Antarctic sea ice proxy IPSO25.

The presence of a di-unsaturated highly branched isoprenoid (HBI) lipid biomarker (diene II) in Southern Ocean sediments has previously been proposed as a proxy measure of palaeo Antarctic sea ice. Here we show that a source of diene II is the sympagic diatom Berkeleya adeliensis Medlin. Furthermore, the propensity for B. adeliensis to flourish in platelet ice is reflected by an offshore downward gradient in diene II concentration in >100 surface sediments from Antarctic coastal and near-coastal environments. Since platelet ice formation is strongly associated with super-cooled freshwater inflow, we further hypothesize that sedimentary diene II provides a potentially sensitive proxy indicator of landfast sea ice influenced by meltwater discharge from nearby glaciers and ice shelves, and re-examination of some previous diene II downcore records supports this hypothesis. The term IPSO25-Ice Proxy for the Southern Ocean with 25 carbon atoms-is proposed as a proxy name for diene II.

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.

Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney.

cDNA complementary to mRNA coding for the beta subunit of dog renal (Na+ + K+)-ATPase has been cloned into lambda gt11 and the nucleotide sequence of the DNA has been determined. The amino acid sequence of the beta subunit polypeptide has also been deduced from the DNA. The mature form of the dog kidney beta subunit contains 302 amino acids with three potential asparagine-linked attachment sites for carbohydrate. The initiation methionine is removed during processing of the polypeptide to its mature form. Although the beta subunit is an integral membrane protein there is no signal sequence for the polypeptide, and hydropathy analysis predicts that the beta subunit polypeptide spans the cell membrane only once. Secondary structure predictions and a model for the structure of the beta subunit are proposed. DNA sequencing of the 5' non-coding region of the mRNA revealed a 200 bp inverted repeat from the coding region. Blot hybridization of a fragment of the beta subunit cDNA identified a single mRNA species of 2.7 kb in dog kidney and several rat tissues. RNA from rat liver was deficient in mRNA that hybridized to the dog kidney beta subunit cDNA, although mRNA that hybridized to an alpha subunit cDNA was detected. RNA from a human hepatoma cell line, HepG2, however, contained comparable levels of mRNA for both the alpha and the beta subunits.

Osteopenia and impaired fracture healing in aged EP4 receptor knockout mice.

The EP4 receptor, one of the subtypes of the prostaglandin E2 (PGE2) receptor, plays a critical role in the anabolic effects of PGE2 on bone. However, its role in the maintenance of bone mass in aged animals and its role in fracture healing is not well known. Our studies addressed these issues by characterizing the skeletal phenotype of aged, EP4 receptor knockout (KO) mice, and by comparing fracture healing in aged KO mice versus wild type (WT) mice. There was no significant difference in body weight and femoral length between KO and WT mice at 15 to 16 months of age. Lower bone mass was seen radiographically in both axial and long bones of KO mice relative to WT mice. Micro-CT images of the distal femurs showed thinner cortices, fewer trabeculae, and a deteriorated trabecular network in KO mice. Total bone content, trabecular content, and cortical content, as assessed by pQCT in the distal femur, were lower in KO mice than WT controls. Histomorphometric measurements showed that trabecular bone volume and bone formation rate were significantly decreased whereas osteoclast number on trabecular surface and eroded surface on endocortical surface were significantly increased in KO mice. These data indicated that deleting the EP4 receptor resulted in an imbalance in bone resorption over formation, leading to a negative bone balance. The lower bone formation rate in EP4 KO mice was primarily due to decreased mineralizing surface, suggesting that the defect in overall bone formation was mainly due to the defect in osteoblastogenesis. Fracture healing was examined in KO and WT mice subjected to a transverse femoral fracture. Callus formation was significantly delayed as evidenced both radiographically and histologically in the fractured femurs of KO mice compared with those of WT mice. KO mice had significant decreases in total callus area, cartilaginous callus area, and bony callus area 2 weeks after fracture. By 4 weeks, complete bony bridging was seen in WT mice but not in KO mice. These data demonstrate that the absence of the EP4 receptor decreases bone mass and impairs fracture healing in aged male mice. Our findings indicate that the EP4 receptor is a positive regulator in the maintenance of bone mass and fracture healing.

Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing.

We present the first indication of a direct relationship between a nuclear and a mitochondrial splicing system. The intron in the precursor of the large, nuclearly coded ribosomal RNA of two species of Tetrahymena possesses all the features of a class of fungal mitochondrial introns. Sequences conserved in mitochondrial introns of different fungal species are also found in the same order in these Tetrahymena nuclear introns, and the intron RNA can be folded to form a secondary structure similar to that proposed for mitochondrial introns by Davies et al. (1982). This "core" secondary structure brings the ends of the intron together. Furthermore, the first intron in the precursor of the large, nuclearly coded rRNA of Physarum polycephalum also has the characteristic conserved sequences and core RNA secondary structure. The limited sequence data available suggest that the intron in the large rRNA of chloroplasts in Chlamydomonas reinhardtii also resembles the mitochondrial introns. Tetrahymena large nuclear rRNA introns also have an internal sequence that can act as an adaptor by pairing with upstream and downstream exon sequences adjacent to the splice junctions to precisely align the splice junctions. These nuclear introns therefore fit the model of the role of intron RNA in the splicing process that was proposed by Davies et al. (1982), suggesting that the mechanisms of splicing may be very similar in these apparently diverse systems. It is therefore probable that the RNA secondary structures for which there is good evidence in the case of mitochondrial introns will be found to form the basis of active site structure and precise alignment in splicing and cyclization of the Tetrahymena intron "ribozyme".

Processing of mitochondrial RNA in Aspergillus nidulans.

Genes for cytochrome oxidase subunit I (oxiA), ATPase subunit 9, NADH dehydrogenase subunit 3 (ndhC) and cytochrome oxidase subunit II (oxiB) are located within a 7.2 kb (1 kb = 10(3) bases or base-pairs) segment of the Aspergillus nidulans mitochondrial genome. Northern hybridization shows that abundant RNA molecules of 4.0, 2.5 and 1.5 kb, each containing copies of two or more genes, are transcribed from this region. The 4.0 kb molecule, which contains copies of each of the four genes but lacks the three oxiA introns, is cleaved at a point just upstream from ndhC to give rise to the 2.5 kb RNA, which contains copies of oxiA and the ATPase subunit 9 gene, and the 1.5 kb RNA, which carries ndhC and oxiB. The ATPase subunit 9 gene, which has no identified function, is therefore transcribed into an abundant RNA. S1 nuclease analysis indicates that there are no additional introns in the amino-terminal region of oxiA and that the 4.0 and 2.5 kb transcripts of this gene have staggered 5' termini, the most upstream of which is adjacent to the 3' end of the histidinyl-tRNA gene. The results suggest that transcription of this genome proceeds via a very limited number of primary transcripts with mature RNAs produced by extensive processing events including tRNA excision. RNA synthesis and processing in A. nidulans mitochondria therefore resembles the events occurring in metazoa rather than yeast.

Network analysis provides insights into evolution of 5S rDNA arrays in Triticum and Aegilops.

We have used network analysis to study gene sequences of the Triticum and Aegilops 5S rDNA arrays, as well as the spacers of the 5S-DNA-A1 and 5S-DNA-2 loci. Network analysis describes relationships between 5S rDNA sequences in a more realistic fashion than conventional tree building because it makes fewer assumptions about the direction of evolution, the extent of sexual isolation, and the pattern of ancestry and descent. The networks show that the 5S rDNA sequences of Triticum and Aegilops species are related in a reticulate manner around principal nodal sequences. The spacer networks have multiple principal nodes of considerable antiquity but the gene network has just one principal node, corresponding to the correct gene sequence. The networks enable orthologous groups of spacer sequences to be identified. When orthologs are compared it is seen that the patterns of intra- and interspecific diversity are similar for both genes and spacers. We propose that 5S rDNA arrays combine sequence conservation with a large store of mutant variations, the number of correct gene copies within an array being the result of neutral processes that act on gene and spacer regions together.

Reliability of DSM-III-R anxiety disorder categories. Using the Anxiety Disorders Interview Schedule-Revised (ADIS-R).

A large reliability study of DSM-III-R anxiety disorders is reported in which outpatients (n = 267) received two independent structured interviews (Anxiety Disorders Interview Schedule-Revised). It is the only reliability study to date in which the final DSM-III-R criteria are used throughout the study. Reliability was assessed for each diagnosis when it was assigned as a principal diagnosis and when it was assigned as either a principal or an additional diagnosis. Excellent reliability was obtained for current principal diagnoses of simple phobia, social phobia, and obsessive-compulsive disorder. Agreement was good for panic disorder when all severity levels of agoraphobic avoidance were combined. Reliability was fair for generalized anxiety disorder. Remaining diagnostic difficulties, particularly in identifying levels of agoraphobic avoidance and in reliably diagnosing generalized anxiety disorder, are discussed in the context of changes in diagnostic criteria that are under consideration for DSM-IV.

Preferential transport of IgA and IgA-immune complexes to bile compared with other external secretions.

When radiolabeled dinitrophenyl-human serum albumin was injected intravenously in mice together with M315 IgA, labeled antigen was specifically transported into bile, but not into saliva, urine, milk or bronchial and intestinal secretions. Ultracentrifugal analysis showed that the antigen transported into bile was intact and partly complexed with IgA. The radioactivity that was present in other secretions regardless of M315 IgA, represented free and degraded fragments of antigen. M315 IgA alone was readily transported into bile, where it was detected at high titer by hemagglutination, but not into other secretions, apart from milk which contained only very low titers. The liver therefore appears to be singularly capable of transporting both free and complexed IgA into its secretion, the bile.

Selective hepatobiliary transport of human polymeric IgA in mice.

Both subclasses of human polymeric IgA (pIgA) were selectively transported from the serum into the bile of mice relative to human IgG or IgM. Removal of human pIgA from serum corresponded to the clearance kinetics shown for murine pIgA. The biliary pIgA was intact as determined by sucrose density gradient ultracentrifugation. This hepatic uptake was specific for the IgA isotype and occurred independently of receptors in the liver specific for glycoproteins that terminate with galactose or mannose moieties. Desialylation of human pIgA resulted in its rapid clearance from serum and subsequent deposition in the liver in a manner similar to most other desialylated serum glycoproteins. The desialylated pIgA present in bile was also an intact molecule; thus the asialoglycoprotein receptor may represent an additional mechanism for the transport of serum pIgA into bile.

Analysis of the hepatobiliary transport of IgA with monoclonal anti-idiotype and anti-allotype antibodies.

The processing and fate of mixed immune complexes is influenced by the antibody isotypes present. The hepatobiliary transport of mixed immune complexes containing the mouse IgA myeloma protein J558 and corresponding monoclonal IgG or IgM anti-J558 idiotype or monoclonal IgG anti-mouse IgA allotype antibodies has been studied. The anti-idiotype or anti-allotype antibodies were radiolabeled and injected into mice with or without mouse polymeric IgA (J558). IgG anti-idiotype antibodies to J558 IgA were selectively transported into bile by J558 IgA. This process occurred with a radiolabeled Fab preparation of the IgG anti-idiotype and was inhibitable with IgA of an irrelevant antigenic specificity. Thus, polymeric IgA influenced the fate of IgA-IgG idiotype-anti-idiotype serum immune complexes. A monoclonal anti-idiotype antibody of the IgM isotype (D8-3) was not selectively transported into bile by itself or as an IgA-IgM complex. A monoclonal IgG antibody (CB5-6) to a mouse allotype determinant in the Fc portion of IgA was not selectively transported into bile. This anti-allotype monoclonal antibody inhibited the hepatobiliary transport of 125I-polymeric J558 IgA and therefore appeared to directly or indirectly block the site in the Fc region of IgA recognized by the hepatic receptor.