Maternal immune and affiliative biomarkers and sensitive parenting mediate the effects of chronic early trauma on child anxiety.

BACKGROUND: Chronic early trauma alters children's stress reactivity and increases the prevalence of anxiety disorders; yet the neuroendocrine and immune mechanisms underpinning this effect are not fully clear. Animal studies indicate that the mother's physiology and behavior mediate offspring stress in a system-specific manner, but few studies tested this external-regulatory maternal role in human children exposed to chronic stress. METHODS: We followed a unique cohort of children exposed to continuous wartime trauma (N = 177; exposed; N = 101, controls; N = 76). At 10 years, maternal and child's salivary immunoglobulin A (s-IgA) and oxytocin (OT), biomarkers of the immune and affiliation systems, were assayed, maternal and child relational behaviors observed, mother and child underwent psychiatric diagnosis, and child anxiety symptoms assessed. RESULTS: War-exposed mothers had higher s-IgA, lower OT, more anxiety symptoms, and their parenting was characterized by reduced sensitivity. Exposed children showed higher s-IgA, more anxiety disorders and post traumatic stress disorder, and more anxiety symptoms. Path analysis model defined three pathways by which maternal physiology and behavior impacted child anxiety; (a) increasing maternal s-IgA, which led to increased child s-IgA, augmenting child anxiety; (b) reducing maternal OT, which linked with diminished child OT and social repertoire; and (c) increasing maternal anxiety, which directly impacted child anxiety. CONCLUSIONS: Our findings, the first to measure immune and affiliation biomarkers in mothers and children, detail their unique and joint effects on children's anxiety in response to stress; highlight the relations between chronic stress, immune activation, and anxiety in children; and describe how processes of biobehavioral synchrony shape children's long-term adaptation.

Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Deregulated expression of homeobox-containing genes, HOXB6, B8, C8, C9, and Cdx-1, in human colon cancer cell lines.

Previously we have demonstrated a reciprocal deregulation of various homeobox genes (HOXB6, B8, C8 and C9 vs Cdx-1) in human colorectal cancer (CRC). In the present study, using RT-PCR, we have investigated the expression pattern of these homeobox genes in various human colon cell lines, representing various stages of colon cancer progression and differentiation. Thus, we have tested polyposis coli Pc/AA adenoma cells, Caco-2, HT-29 and LS174T adenocarcinoma cell lines. All cell lines, except LS174T, demonstrated a pattern of deregulated homeobox gene expression which resembled that of CRC. In contrast, the pattern of expression of these genes in the highly oncogenic LS174T cells, as well as in Caco-2 cells transfected with activated Ha-ras or Polyoma middle T oncogene, resembled that of the normal mucosa. The reciprocal deregulation of HOX and Cdx-1 genes in CRC and in CRC-derived cell lines suggests a possible role in human CRC development.

Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness.

The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.

Human frizzled 1 interacts with transforming Wnts to transduce a TCF dependent transcriptional response.

The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.

Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling.

Frizzled related proteins (FRPs) comprise a family of secreted molecules that contain an N-terminal cysteine-rich domain (CRD) highly similar to the CRDs of the frizzled family of membrane-anchored Wnt receptors. FRPs have been shown to interact with Wnt proteins and antagonize Wnt signaling in a Xenopus developmental model. We demonstrated that FRP antagonizes the Wnt-induced increase in uncomplexed beta-catenin in both transient cotransfection and stable transformation models, where Wnt-induced morphological alterations are inhibited as well. We showed further that FRP inhibits Wnt signaling in a paracrine mode using a T-cell factor luciferase reporter to measure Wnt function. Investigation of the mechanisms responsible for FRP inhibition revealed that FRP forms complexes with WNT-1 or WNT-2 through its CRD domain. Transfection analysis with FRPs containing different tags revealed that FRP itself forms complexes and that this ability is conferred by its CRD domain. Finally, we demonstrated by cotransfection that FRP forms complexes with a prototype frizzled. All of these findings are consistent with a model by which FRP inhibits Wnt signaling through interactions with Wnt and/or formation of nonfunctional complexes with the frizzled receptor.

cDNA excision in stable retroviral cDNA transfectants is prevented by R removal.

The present study provides evidence on the occurrence of DNA rearrangement between the redundant 5' and 3' R domains of equine infectious anemia virus (EIAV) Tat cDNA. This was correlated with a gradual loss of cDNA copy number concomitantly with a decrease in gene expression. Removal of the 5' RU5 abolished rearrangement and stabilized Tat expression in EIAV tat cDNA transfectants. Our data suggest that prior removal of the 5' R from cloned retroviral cDNAs can impede DNA rearrangement, thus preventing cDNA excision that frequently occurs and hinders permanent expression of retroviral cDNAs in stable transfectants.

The Tat protein of equine infectious anemia virus (EIAV) activates cellular gene expression by read-through transcription.

The Tat protein of equine infectious anemia virus, EIAV, was shown to augment viral gene expression, presumably through interaction with the Tat responsive element, TAR. Recently, cell-free polyadenylation assays suggested that perturbation of the EIAV TAR secondary structure diminished polyadenylation efficiency. The present study indicates that the EIAV TAR regulates the efficiency of the 3'-end processing of viral RNA also in transfected cells. Moreover, our data suggest that the provision of the EIAV Tat protein in trans potentiates read-through transcription through the 3' viral long terminal repeat (3' LTR), thus suggesting activation of downstream-located cellular genes.

Characterization of Wnt-1 and Wnt-2 induced growth alterations and signaling pathways in NIH3T3 fibroblasts.

Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness. However, unlike oncogenes such as PDGFB or ras, Wnt-1 and -2 failed to induce detectable transformed foci following transfection, and stable NIH3T3 transfectants lacked tumor forming capacity. Wnt-1 and -2 transfectants exhibited increased uncomplexed, cytosolic beta-catenin, which was not observed with PDGFB, ras or erbB2 transfectants. In transient transfection, Wnt-1 and -2 induced a rapid increase in cytosolic beta-catenin but no detectable increase in the phosphorylated activated forms of MAP kinase. In contrast, ras was a potent activator of MAP kinase but had no effect on free beta-catenin levels. These findings establish that both Wnt signaling and pattern of growth alterations differ from those of oncogenes which activate proliferative signaling pathways in NIH3T3 cells.

Productive replication of caprine arthritis-encephalitis virus is associated with induction of apoptosis.

Caprine arthritis-encephalitis virus (CAEV), an ungulate lentivirus, causes a natural infection in goats. The present report demonstrates that in vitro, CAEV infection is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearance of apoptotic bodies. The presence of DNA fragments was documented by the appearance of a DNA 'ladder' in agarose gel electrophoresis, as well as by in situ end-labelling of DNA ends. In addition, flow cytometric analyses revealed the presence of a hypodiploid peak that gradually appeared as virus infection progressed.

Human colorectal carcinogenesis is associated with deregulation of homeobox gene expression.

In the present study, the possible involvement of homeobox-containing genes in colorectal cancer (CRC) development was investigated. Using a stepwise screening approach and RT-PCR, we have demonstrated that the human HOXB6, B8, C8 and C9 are overexpressed at various stages of CRC. In contrast, all CRC cases exhibited a marked decrease in the homeodomain-containing Cdx1 gene expression. Recent data which suggest a regulatory link between HOXB8 and several tumor suppressor genes, such as DCC, APC, and TGF beta, sustain a possible implication of homeobox genes in colon carcinogenesis. Moreover, our data showing a decrease in Cdx1 expression are consistent with the notion that genes functioning in the establishment and maintenance of the intestinal epithelium might, upon deregulation, disturb the normal control of cellular proliferation, differentiation, and death, thus leading to cancer development.

The turkey c-rap1A proto-oncogene is expressed via two distinct promoters.

The turkey c-K-ras(B) transcript and two species of the turkey rap1A transcripts transcribed from two distinct promoters were isolated from a turkey spleen cDNA library. Turkey K-Ras and Rap1A proteins shared extensive amino acid (aa) sequence relatedness throughout their major functional domains: the four GTP-binding domains, the effector region and the C-terminal CAAX box. However, they diverged significantly in their intervening regions. In contrast, almost complete identity in the aa composition was exhibited between turkey K-Ras and Rap1A and their human homologues. The complete conservation that exists between turkey and human Rap1A, also along the polybasic C-terminal domain as opposed to Rap1B, suggests the functional relevance of these divergent residues in specifying the distinct biological functions of these two closely related proteins.

Structural studies of the equine infectious anemia virus trans-activator protein.

Trans-activator (tat) proteins are necessary components for the completion of the T replication cycle of lentiviruses. The three-dimensional structure of the equine infectious anemia virus (EIAV) tat protein (e-tat) was studied with CD spectroscopy, NMR spectroscopy, and restrained molecular-dynamics calculations. No stable elements of regular secondary structure were detected, but the sequence regions responsible for nucleic acid binding showed helix-forming tendency, e-tat exhibits a flexible tertiary structure, and only the amino acids comprising the core sequence region form a well-defined tertiary fold. The three-dimensional structure allows discussion of biochemical data as well as data from molecular biological investigations of lentiviral tat proteins.

Two species of Rev proteins, with distinct N termini, are expressed by caprine arthritis encephalitis virus.

Several cDNA clones representing alternatively spliced Rev-specific transcripts were isolated from a cDNA library prepared from Himalayan tahr cells infected with caprine arthritis encephalitis virus (CAEV). We previously characterized two rev-like cDNA species, d1 and d2, and a tat e1 cDNA containing the rev coding sequence downstream to the tat. In these cDNAs, the rev coding domain derives its amino terminus from the N terminus of env, which is spliced to the 3' open reading frame encoding the putative Rev protein. In this study, we report the genetic structure of a fourth rev-like cDNA (designated g1), which lacks the 5' env-derived sequences. All of these rev transcripts, including cDNA g1, increased the level of chloramphenicol acetyltransferase expression when cotransfected with a reporter plasmid containing the CAEV Rev-response element-spanning region downstream of the cat coding sequences. Western blot (immunoblot) analysis showed that each transfected cDNA species gave rise to a 16-kDa protein lacking env-encoded amino-terminal epitopes. In contrast, CAEV-infected Himalayan tahr cells expressed only a 20-kDa protein, whose N terminus, in contrast, is derived from the env. Moreover, only the 20-kDa protein was also detected in the mature CAEV virions. These observations suggest that the transcripts d1, d2, and e1 can potentially, in appropriate cellular context, encode two Rev isoforms differing in their N termini, whereas the g1 transcript encodes only the 16-kDa species. Elucidation of the significance of the 16-kDa Rev protein in CAEV biology must await further studies.

Evidence for the involvement of the Wnt 2 gene in human colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent cancers in humans. It develops via a multistage process involving alterations of both protooncogenes and tumor suppressor genes. In the present report we determined the level of expression of several Wnt genes in CRC by RT-PCR and direct sequencing. While Wnt-1 was not detectably expressed in any colonic tissues, Wnt-5a gene was efficiently expressed both in nontumorous as well as in colonic tumor tissues. In contrast, the Wnt-2 gene, which was expressed at low levels in normal colon, exhibited overexpression in all tumor tissue samples at the different Dukes' stages of CRC progression, including premalignant polyps and liver metastases. Overexpression of the Wnt-2 gene occurred also in other digestive neoplasms such as gastric and esophageal carcinomas, as well as in diverticulitis associated with stenosis or pseudo-tumor.

Identification of sequences in the long terminal repeat of the lymphoproliferative disease virus required for efficient transcription.

We analyzed the long terminal repeat (LTR) of the lymphoproliferative disease virus of turkeys for sequences that influence its promoter activity by using the chloramphenicol acetyltransferase assay. A series of LTR deletion mutants and recombinants between LTR and simian virus 40 regulatory sequences were used for these studies. Through transfection experiments, we identified a negative regulatory element residing at the 5' end of the U3. The two imperfect direct repeats (DRs) located at nt - 170 to - 125 upstream of the RNA transcription site were identified as enhancer elements which could stimulate transcription of a heterologous promoter in an orientation independent manner. Specific interaction of nuclear factors with the DRs element was identified. The two DRs contain cArg motifs which are suggested to play a role in tissue specific expression of several cellular genes.

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus.

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10(11) bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by 'scrape loading'. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.

Trifluoroethanol stabilizes a helix-turn-helix motif in equine infectious-anemia-virus trans-activator protein.

The solution structure of the 75-amino-acid trans-activator (Tat) protein of the equine infectious-anemia virus in trifluoroethanol-containing solution was determined by two-dimensional and three-dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear-Over-hauser-enhancement distance restraints, and restrained molecular-dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol-free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA-binding region of the protein showed well-defined alpha-helical secondary structure in trifluoroethanol-containing solution. The helical regions comprise those parts of the molecule whose helix-forming tendencies were noted earlier in trifluoroethanol-free solution. Two helices (Gln38-Arg43 and Asp48-Ala64) are connected by a tight type-II turn centered at the strictly conserved Gly46 leading to a helix-turn-helix motif in the core and basic region of the protein. A third helix (Thr9-Asn13) is located in the less well defined N-terminal part of the protein. These observations may support the notion that the protein adopts a helical structure in the RNA-binding region on complex formation. Although the secondary-structure elements become better defined in trifluoroethanol-containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol-containing solution in general.