Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

Developmental study of Müller cells in the rat retina using a new monoclonal antibody, RT10F7.

We produced the monoclonal antibody RT10F7, characterized its antigenic specificity and expression in the adult and developing retina, in cultured retinal cells and in other parts of the central nervous system. In metabolically-labelled retinal cultures RT10F7 immunoprecipitated a protein of approximately 36,000 mol. wt. In the adult, RT10F7 stained endfeet of Müller cells in the ganglion cell layer, four horizontal bands in the inner plexiform layer, and radial fibres in the outer plexiform layer which terminated at the outer limiting membrane. In the inner nuclear layer, most somata were underlined by Müller processes that wrapped around them, but some cell bodies were immunoreactive for RT10F7 in the cytoplasm. During development, postnatal day 21 was the first age at which the adult pattern of immunoreactivity was present, although a fourth band in the inner plexiform layer was less clear than for the adult. By 14 and eight days after birth, the pattern of RT10F7 immunoreactivity approximated that of the adult; however, only three bands and one band were present, respectively, in the inner plexiform layer. At earlier ages, postnatal days 4, 1 and embryonic ages 19 and 15, the monoclonal antibody stained Müller cell endfeet and radial fibres, from the inner plexiform layer through the neuroblastic layer to the outer limiting membrane. At these ages, the immunoreactivity was more prominent at the level of Müller cell endfeet. The monoclonal antibody stained glia in preparations of dissociated retinal cells maintained in culture but not astrocytes or oligodendrocytes from optic nerve cultures. In brain sections, tanycytes exhibited RT10F7 immunoreactivity. The monoclonal antibody RT10F7 recognized a specific cell type in the retina, the Müller cell. In the adult and developing retina, RT10F7 recognized an antigen that is present primarily in Müller cell processes. This feature allowed us to follow the maturation of the Müller cell and correlate it with developmental events in the retina. RT10F7 is a specific marker for Müller cells in vivo and in vitro and may be useful for studies of function of Müller cells after ablation or after injuries that are known to activate Müller cells.

Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin.

Signaling by the Wnt family of extracellular proteins is critical in a variety of developmental processes in which cell and tissue polarity are established [1-5]. Wnt signal transduction has been studied mostly by the genetic approach in Drosophila and Caenorhabditis elegans [1,2,5], but the biochemical mechanisms involved remain to be elucidated. The Wnt pathway also operates during axis determination in vertebrates [3,5]. Frizzled receptors transduce a signal to Dishevelled, leading to inactivation of glycogen synthase kinase 3 (GSK3) and regulation of gene expression by the complex of beta-catenin with LEF/TCF (lymphocyte enhancer factor/T-cell factor) transcription factors [3,5]. Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates [6]. Here, we demonstrate that axin is associated with GSK3 in the Xenopus embryo and we localize the GSK3-binding domain to a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). We also find that wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos.

[The regulatory role of natural antibodies and prostaglandin F2 alpha in the course of a myocardial infarct]. / Reguliatornaia rol' estestvennykh antitel i prostaglandinov F2 al'fa v techenii infarkta miokarda.

Concentration of PGF2 alpha were correlated to those of antiprostaglandin antibodies IgM and IgG to this mediator in donors and myocardial infarction patients. In donors correlation between PGF2 alpha concentration and IgG antibody levels is positive. In uneventful myocardial infarction relevant correlation partially recovered at the expense of IgM antibodies. This is not true for complicated myocardial infarction.

[Natural anti-PGF2alpha and anti-PGE2 antibodies in ischemic heart disease and hypertension]. / Estestvennye antitela k prostaglandinam F2alpha i E2 pri ishemicheskoi bolezni serdtsa i gipertonicheskoi bolezni.

The important role played by prostaglandins in the pathogenesis of coronary disease and essential hypertension is well known. The authors have attempted to reveal a relationship between blood levels of natural autoantibodies to PGF2 alpha and PGE2 and specific features of coronary disease and essential hypertension course and complications. A total of 87 subjects were examined, 23 of these--normal controls, 25 patients with myocardial infarction, 22 with angina pectoris, and 17 with essential hypertension. Solid-phase enzyme immunoassay was employed to detect anti-PGF2 alpha and anti-PGE2 antibodies. These antibodies were found in normal subjects, coronary patients and hypertensives. Blood levels of these antibodies correlated with some complications of coronary disease and essential hypertension. These results permit a hypothesis that the pathogenetic physiologic role of the detected natural antibodies to prostaglandins consists in their defense homeostatic function.

[Comparative analysis of the levels of anti-prostaglandin F2 alpha antibodies in the normal state and in cardiovascular diseases]. / Sravnitel'nyi analiz soderzhaniia autoantitel protiv prostaglandina F2 alpha v norme i pri serdechno-sosudistykh zabolevaniiakh.

The ELISA test was performed on sera of normal donors and patients with myocardial infarction (MI) and essential hypertension (EH) to detect autoantibodies against prostaglandin F2 alpha (a-PGF2 alpha). The a-PGF2 alpha level was much higher after immune complexes dissociation. Baseline a-PGF2 alpha was elevated in EH patients with a sudden rise in blood pressure, and in MI patients without hypertension and unstable angina. MI patients with bradyarrhythmias also had elevated a-PGF2 alpha levels. Baseline a-PGF2 alpha was rather low in the sera of donors, chronic EH patients with hypertension and unstable angina, suggesting that a-PGF2 alpha has a physiological role to play.

[Humoral autoimmune reactions directed against autoantigens of the H-2 histocompatibility system in mice with developing Rauscher leukemia]. / Gumoral'nye autoimmunnye reaktsii myshei s razvivaiushchimsia leikozom Raushera, napravlennye protiv sobstvennykh antigenov gistosovmestimosti sistemy H-2.

Antibodies exhibiting a selective anti-H-2 haplotype reaction with thymic and splenic lymphocytes of intact mice were found in the sera of mice of the strains BALB/c, C57Bl/6, AKR and BDF1 with the developing Rauscher leukemia by applying the membrane immunofluorescent and complement-dependent cytotoxicity techniques in vitro. Monoclonal antibodies against H-2 IAd and H-2 IAk and sorption tests using lymphocytes of the congenic-resistant strains as target cells were used to show that the aforementioned antibodies acted against autoantigens which are the products of the genes of the I-subregion of the H-2 histocompatibility complex.

[Autogenic anti-idiotypic suppression of autoimmune reactions--a new approach to immunotherapy of experimental leukemia]. / Autogennoe antiidiotipicheskoe podavlenie autoimmunnykh reaktsii--novyi podkhod k immunoterapii eksperimental'nykh leikozov.

Experimental Rauscher's virus erythroleukemia (RVE) was employed to study the immunologic mechanisms by which leukemia develops. Experiments were performed on inbred mice, genetically opposite to the disease induction, namely on highly sensitive BALB/c, resistant C57BL/6 and moderately sensitive BDFI animals. It is shown that RVE resistance is an immunologic phenomenon that results from the functioning of the antileukemic immune defence (ALID) aimed against the tumor-specific antigenic complex. Suppression of the ALID stems from autosuppression of the H-2 complex of the histocompatibility antigen system of T suppressors, which leads to the development of the onco-specific complex of autoimmune responses (OSCAR) and to the obligate development of RVE. The recovery of the ALID with OSCAR suppression and RVE regression is a consequence of the development of strictly specific antiidiotypic immune responses (antibodies AIT-anti-OSCAR). It is demonstrated that both passive administration of AIT-anti-OSCAR and induction of their active synthesis brings about a remission of RVE.