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PubMed2003

Comparative effects of conjugated and deconjugated isomeric enones on the transannular Diels-Alder reaction.

Two isomeric cyclic trienones 2 and 3 (with four methyl esters at positions 3 and 10) underwent transannular Diels-Alder (TADA) reaction at very different temperatures. This drastic difference could be traced to the capacity of the enone dienophile to be conjugated or unconjugated at the transition state. Molecular modeling could confirm this explanation. The calculated enone proved to be very distorted in transition state ts2, and the TADA reaction temperature was accordingly much higher than the one corresponding to ts3 in which the enone was flat. [reaction: see text]

Journal Article↗
PubMed2003

Tight-adherence genes of Actinobacillus actinomycetemcomitans are required for virulence in a rat model.

Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that has been associated with localized aggressive periodontitis and infections of the heart, brain, and urinary tract. Wild-type clinical isolates have the remarkable ability to adhere tenaciously and nonspecifically to solid surfaces such as glass, plastic, and hydroxyapatite. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which consists of 14 genes (flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG). All but 2 of the genes have been shown to be required for the secretion and assembly of long, bundled Flp1 fibrils. To test whether the tad locus is required for colonization and disease, we developed a rat model for periodontal disease. To mimic the natural route of infection, Sprague-Dawley rats were inoculated orally by adding bacteria directly to their food for 8 days. After inoculation with wild-type or mutant strains defective in adherence (flp-1 and tadA), the rats were assessed for colonization of the oral cavity and pathogenesis. Wild-type A. actinomycetemcomitans was able to colonize and persist for at least 12 weeks in the oral cavity, elicit a humoral immune response, and cause significant bone loss in rats. In contrast, rats fed flp-1 or tadA mutant strains showed no bone loss and their immune responses were indistinguishable from those of the uninoculated controls. These results demonstrate the critical importance of the tad locus in the colonization and pathogenesis of A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed2000

Normal fertilization occurs with eggs lacking the integrin alpha6beta1 and is CD9-dependent.

Previous results, based on inhibition of fertilization by an anti-alpha6 integrin mAb (GoH3), suggest that the alpha6beta1 integrin on mouse eggs functions as the receptor for sperm (Almeida, E.A., A.P. Huovila, A.E. Sutherland, L.E. Stephens, P.G. Calarco, L. M. Shaw, A.M. Mercurio, A. Sonnenberg, P. Primakoff, D.G. Myles, and J.M. White. 1995. Cell. 81:1095-1104). Because the egg surface tetraspanin CD9 is essential for gamete fusion (Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Suzuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324) and CD9 is known to associate with integrins, recent models of gamete fusion have posited that egg CD9 acts in association with alpha6beta1 in fusion (Chen, M.S., K.S. Tung, S.A. Coonrod, Y. Takahashi, D. Bigler, A. Chang, Y. Yamashita, P.W. Kincade, J.C. Herr, and J.M. White. 1999. Proc. Natl. Acad. Sci. USA. 96:11830-11835; Kaji, K., S. Oda, T. Shikano, T. Ohnuki, Y. Uematsu, J. Sakagami, N. Tada, S. Miyazaki, and A. Kudo. 2000. Nat. Genet. 24:279-282; Le Naour, F., E. Rubinstein, C. Jasmin, M. Prenant, and C. Boucheix. 2000. Science. 287:319-321; Miyado, K., G. Yamada, S. Yamada, H. Hasuwa, Y. Nakamura, F. Ryu, K. Su- zuki, K. Kosai, K. Inoue, A. Ogura, M. Okabe, and E. Mekada. 2000. Science. 287:321-324). Using eggs from cultured ovaries of mice lacking the alpha6 integrin subunit, we found that the fertilization rate, fertilization index, and sperm binding were not impaired compared with wild-type or heterozygous controls. Furthermore, a reexamination of antibody inhibition, using an assay that better simulates in vivo fertilization conditions, revealed no inhibition of fusion by the GoH3 mAb. We also found that an anti-CD9 mAb completely blocks sperm fusion with either wild-type eggs or eggs lacking alpha6beta1. Based on these results, we conclude that the alpha6beta1 integrin is not essential for sperm-egg fusion, and we suggest a new model in which CD9 acts by itself, or interacts with egg protein(s) other than alpha6beta1, to function in sperm-egg fusion.

Animals↗
PubMed1977

Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT) II. Presence of I-J determinants on the GT-suppressive factor.

The responses to the synthetic antigens, L-glutamic acid(60)-L- alanine(30)-L-tyrosine(10) (GAT) and L-glutamic acid(50)-L-tyrosine(50) (GT) are controlled by genes in the I region of the mouse H-2 complex (1-3). Preimmunization of the mice bearing the H-2(p,q,s) nonresponder haplotypes with GAT stimulates the development of suppressor T cells that inhibit in vivo or in vitro antibody responses to GAT complexed to the immunogenic carrier, methylated bovine serum albumin (GAT-MBSA) (4). The copolymer GT is not immunogenic in any inbred mouse strain tested, and has a suppressive effect on the antibody responses to GT-MBSA in mouse strains bearing the H-2(d,f,k,s) haplotypes; suppressor T cells have been demonstrated to be responsible for specific GT suppression (3). We have obtained specific suppressive extracts from thymus and spleen cells of GAT-or GT-primed suppressor strains (5,6). The specific suppressive T-cell factors in the active extracts have been characterized (6,7) and appear similar to the carrier-specific suppressor factor described by Tada and Taniguchi (8). These products belong to a family of newly identified molecules coded for by the I region of the H-2 complex with affinity for antigen and helper (9,10) or suppressive (5-8) regulatory activity on the immune response. Recently, Tada et al. have reported that the keyhole limpet hemocyanin (KLH)-specific suppressor factor is coded for by the I-J subregion of the H-2 complex (11). We now demonstrate also that a GT-specific suppressor factor extracted from the spleens and thymuses of B10.BR (H-2(k)) mice bears determinants controlled by the I-J subregion of the H-2 complex.

Alanine↗
PubMed2026

damidBind: an R/bioconductor package for differential DamID analysis and data exploration.

SUMMARY: DamID, and its cell-type specific adaptations, including Targeted DamID (TaDa) and Chromatin Accessibility TaDa (CATaDa), are now widely-adopted as techniques for the genome-wide profiling of DNA binding proteins. Despite this popularity, no dedicated software solution exists for identifying differentially bound or accessible loci, or differentially transcribed genes, between cell types using DamID. The R/Bioconductor package damidBind provides these functions, allowing an end-user to move from processed binding profiles to identifying differentially-bound loci in a reproducible, statistically appropriate and straightforward workflow. AVAILABILITY AND IMPLEMENTATION: damidBind is an open-source R/Bioconductor package and freely available from Bioconductor at https://bioconductor.org/packages/damidBind/, and from GitHub at https://github.com/marshall-lab/damidBind. It is released under the GPLv3 licence.

Software↗
PubMed1989

Oncogene-associated tumor antigens as targets for immunotherapy.

Cellular antigens encoded by tumor viruses and some antigens encoded by cellular oncogenes offer advantages as targets for immunotherapy by being inextricably associated with the neoplastic phenotype. For example, monoclonal antibodies (MAb) specific for an antigen encoded by the neu oncogene have a direct inhibitory effect on proliferation of antigen-positive tumor cells. Many of the oncogene-encoded cell surface molecules are growth factor receptors, as are some tumor-associated differentiation antigens (TADAs). Therefore, it is not surprising that their level of cancer specificity is similar. There have been some promising findings from using TADAs as targets for various forms of immunotherapy, and one would expect the results to further improve by targeting to molecules that are more directly involved in cell proliferation and/or in maintaining the malignant state.

Animals↗
PubMed2003

Haemophilus ducreyi requires an intact flp gene cluster for virulence in humans.

An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.

Adult↗
PubMed2006

[Comparative study for the methods on measuring intracranial hemorrhage volume].

OBJECTIVE: To explore a method of measuring intracranial hemorrhage to be applied in clinical forensic science. METHODS: The accurace of the methods of Tada formula, Stereology and Software boundary to measure intracranial hemorrhage and their practicability were compared. RESULTS: The measured results of Stereology and Software boundary are precise, but the irregularity haematoma volume measured by the method of Tada formula has a high error value. CONCLUSION: The method of stereolgy is the best in measuring intracranial hemorrhage for clinical forensic science.

Adolescent↗
PubMedDate not supplied

Serum adenosine deaminase activity and its isoenzyme pattern in patients with systemic lupus erythematosus.

OBJECTIVE: To study the correlation between serum adenosine deaminase (ADA) activity and clinical activity in patients with systemic lupus erythematosus (SLE). The patterns of two ADA isoenzymes, ADA1 and ADA2, were also analysed in healthy controls and patients to determine the source of increased ADA activity in patients. METHODS: Total serum ADA activity (tADA) was measured spectrophotometrically. The isoenzyme pattern of ADA was analysed by the HPLC method using a specific inhibitor of ADA1, erytro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Disease activity was assessed using the European Consensus Lupus Activity Measurement index (ECLAM). RESULTS: Serum tADA activity was significantly increased in patients compared to healthy controls (mean +/- SD; 476.9 +/- 145.3 vs 254.0 +/- 98.9 ncat/L, p < 0.001). The isoenzyme analyses showed that the increased total ADA activity in the patients was mainly due to increased ADA2 activity (371.3 +/- 154.8 vs 214.2 +/- 47.9 ncat/L in healthy controls, p < 0.001). The mean values for ADA1 activity in the patients (64.6 +/- 37.9 ncat/L) and healthy controls (69.2 +/- 26.9 ncat/L) were similar. A strong correlation was found between serum ADA activity and disease activity as measured by ECLAM (Spearman's rank correlation coefficient 0.74, p < 0.0001, linear regression coefficient 0.68, p < 0.01). CONCLUSION: Serum ADA activity is closely associated with SLE activity and appears to be useful as a new disease activity parameter of SLE.

Adenosine Deaminase↗
PubMed1994

Thyroid hormone enhances Ca2+ pumping activity of the cardiac sarcoplasmic reticulum by increasing Ca2+ ATPase and decreasing phospholamban expression.

Phospholamban is a putative suppressor of the Ca2+ ATPase of the cardiac sarcoplasmic reticulum. The level of mRNA encoding the Ca2+ ATPase has been shown to be increased, whereas the phospholamban mRNA level to be decreased in the ventricles obtained from hyperthyroid rabbits [Nagai R, Zarain-Herzberg A. Brandl CJ, Fujii J. Tada M. MacLennan DH, Alpert NR, Periasamy M. (1989) Proc Natl Acad Sci USA 86: 2966-2970]. The present study was designed to examine whether these effects of thyroid hormone on the expression of the Ca2+ ATPase and phospholamban are exerted directly on cardiac myocytes and whether the resultant incoordinate expression of these proteins alters Ca2+ pumping activity. We studied the levels of phospholamban and Ca2+ ATPase mRNA in primary isolated neonatal rat myocardial cells incubated with triiodothyronine (T3) for 3-48 h and the Ca2+ uptake activity of the microsomes prepared from the cells. Northern blot analysis showed that T3 decreased phospholamban mRNA levels to about a half of control in 24 h. On the other hand, Ca2+ ATPase mRNA gradually increased with time. EC50 for phospholamban mRNA expression was 2.5 x 10(-10) M which was approximately 10 times higher than that for the Ca2+ ATPase. T3 increased Vmax of Ca2+ uptake with the significant reduction of K0.5 for Ca2+ (0.40 +/- 0.02 microM for control v 0.31 +/- 0.02 microM for T3-treated vesicles), indicating that thyroid hormone stimulates Ca2+ pumping activity not only by increasing the Ca2+ ATPase but also decreasing phospholamban. These results suggested that phospholamban regulates the Ca2+ ATPase in dual modes; in short time range, by decreasing the affinity of the Ca2+ ATPase for Ca2+ by phosphorylation of phospholamban with cAMP-dependent protein kinase, and in long time range, by changing the molecular ratio between the two proteins through the regulation of gene expression.

Animals↗
PubMed1998

Phospholamban domain Ib mutations influence functional interactions with the Ca2+-ATPase isoform of cardiac sarcoplasmic reticulum.

Alanine-scanning mutagenesis of amino acids 21-30, forming cytoplasmic domain Ib in phospholamban (PLN), revealed that mutation to Ala of Asn27, Gln29, and Asn30 results in gain of inhibitory function. In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M. , and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), gain of function in PLN transmembrane domain II mutants was correlated with pentamer destabilization, leading to proposals that the PLN monomer is the active inhibitory species, that dissociation of the PLN pentamer is one determinant of PLN inhibitory function and that dissociation of the PLN.cardiac sarco(endo)plasmic Ca2+-ATPase isoform (SERCA2a) complex is a second determinant. Because each of the new domain Ib mutants contained a normal ratio of pentamer to monomer in SDS-polyacrylamide gel electrophoresis, gain of function must have resulted from mechanisms other than destabilization of pentameric structure. Evidence that domain Ib and domain II mutants act through different sites and different mechanisms was provided by a monomeric double mutant, N30A/I40A, in which the enhanced inhibitory function of each single mutant was additive. Evidence for an alteration in stability of the PLN/SERCA2a heterodimer was obtained in a study of double mutant N27A/N34A in which inhibitory function was regained by combining a gain of function, domain Ib mutation with a loss of function domain II mutation. These results support the proposal that PLN inhibition of SERCA2a involves, first, depolymerization of PLN and, second, the formation of inhibitory interactions between monomeric PLN and SERCA2a.

Animals↗
PubMed1999

Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites.

In an earlier study (Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1997) J. Biol. Chem. 272, 15061-15064), mutation of amino acids on one face of the phospholamban (PLN) transmembrane helix led to loss of PLN inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) molecules. This helical face was proposed to form a site of PLN interaction with a transmembrane helix in SERCA molecules. To determine whether predicted transmembrane helices M4, M5, M6, or M8 in SERCA1a interact with PLN, SERCA1a mutants were co-expressed with wild-type PLN and effects on Ca(2+) dependence of Ca(2+) transport were measured. Wild-type inhibitory interactions shifted apparent Ca(2+) affinity of SERCA1a by an average of -0.34 pCa units, but four of the seven mutations in M4 led to a more inhibitory shift in apparent Ca(2+) affinity, averaging -0.53 pCa units. Seven mutations in M5 led to an average shift of -0.32 pCa units and seven mutations in M8 led to an average shift of -0.30 pCa units. Among 11 mutations in M6, 1, Q791A, increased the inhibitory shift (-0.59 pCa units) and 5, V795A (-0.11), L802A (-0.07), L802V (-0.04), T805A (-0.11), and F809A (-0.12), reduced the inhibitory shift, consistent with the view that Val(795), Leu(802), Thr(805), and Phe(809), located on one face of a predicted M6 helix, form a site in SERCA1a for interaction with PLN. Those mutations in M4, M6, or M8 of SERCA1a that enhanced PLN inhibitory function did not enhance PLN physical association with SERCA1a, but mutants V795A and L802A in M6, which decreased PLN inhibitory function, decreased physical association, as measured by co-immunoprecipitation. In related studies, those PLN mutants that gained inhibitory function also increased levels of co-immunoprecipitation of wild-type SERCA1a and those that lost inhibitory function also reduced association, correlating functional interaction sites with physical interaction sites. Thus, both functional and physical data confirm that PLN interacts with M6 SERCA1a.

Biological Transport↗
PubMed2000

Physical interactions between phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPases are dissociated by elevated Ca2+, but not by phospholamban phosphorylation, vanadate, or thapsigargin, and are enhanced by ATP.

Previous co-immunoprecipitation studies (Asahi, M., Kimura, Y., Kurzydlowski, K., Tada, M., and MacLennan, D. H. (1999) J. Biol. Chem. 274, 32855-32862) revealed that physical interactions between phospholamban (PLN) and the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA1a) were retained, even with PLN monoclonal antibody 1D11 bound to an epitope lying between PLN residues 7 and 17. Because the 1D11 antibody relieves inhibitory interaction between the two proteins, it was of interest to determine whether PLN phosphorylation or elevation of Ca(2+), which also relieves inhibitory interactions between PLN and SERCA, would disrupt physical interactions. Co-immunoprecipitation was measured in the presence of increasing concentrations of Ca(2+) or after phosphorylation of PLN by protein kinase A. Physical interactions were dissociated by elevated Ca(2+) but not by PLN phosphorylation. The addition of ATP enhanced interactions between PLN and SERCA. The further addition of vanadate and thapsigargin, both of which stabilize the E(2) conformation, did not diminish binding of PLN to SERCA. These data suggest that physical interactions between PLN and SERCA are stable when SERCA is in the Ca(2+)-free E(2) conformation but not when it is in the E(1) conformation and that phosphorylation of PLN does not dissociate physical interactions between PLN and SERCA.

Adenosine Triphosphate↗
PubMed2006

Association of Gln222Arg polymorphism in the deoxyribonuclease I (DNase I) gene with myocardial infarction in Japanese patients.

AIMS: We have recently reported that serum deoxyribonuclease I (DNase I) activity, which may be involved in apoptosis, increases abruptly in the early phase of acute myocardial infarction (MI) [Kawai Y, Yoshida M, Arakawa K, Kumamoto T, Morikawa N, Masamura K, Tada H, Ito S, Hoshizaki H, Oshima S, Taniguchi K, Terasawa H, Miyamori I, Kishi K, Yasuda T. Diagnostic use of serum deoxyribonuclease I activity as a novel early-phase marker in acute myocardial infarction. Circulation 2004;109:2398-2400]. Death of vascular smooth muscle cells, in part because of apoptosis, is postulated to heighten susceptibility to disruption of vulnerable plaque, resulting in onset of MI. The present study evaluated the possibility that Gln222Arg polymorphism of the DNase I gene may be one of the factors involved in predisposition to MI. METHODS AND RESULTS: We assessed 611 Japanese patients: 311 with MI and 300 with stable angina pectoris (AP). Three common phenotypes determined by two common codominant alleles, DNASE1*1 and *2, whose corresponding gene products exhibit different properties, were found in these patient groups. The prevalence of DNASE1*2 was significantly higher in patients with MI than in those with AP (0.543 vs. 0.428, P < 0.001), being confirmed by phenotyping of the second study population. Multiple logistic regression analysis showed that the odds ratio of DNASE1*2 was 1.51 [95% confidence interval (CI) 1.04-2.18]. The association of the DNASE1*2 allele with MI was statistically significant, being independent of other conventional risk factors. CONCLUSION: Our data demonstrate that Gln222Arg polymorphism in the DNase I gene is associated with MI in the Japanese patients.

Aged↗
PubMed1990

Monoclonal antibodies to disialogangliosides: characterization of antibody-mediated cytotoxicity against human melanoma and neuroblastoma cells in vitro.

We previously reported the binding specificities of two anti-ganglioside GD2 murine monoclonal antibodies (MAbs), A1-425 and A1-267, both of which are of IgG3 isotype. A1-425 reacts specifically with ganglioside GD2, whereas A1-267 binds preferentially to GD2 but also reacts with GD3 [Tai, T., Kawashima, I., Tada, N., & Dairiki, K. (1988) J. Biochem. 103, 682-687]. In this paper, they were used for comparative analyses of antibody-mediated cytotoxicity, i.e., antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human melanoma and neuroblastoma cell lines. Melanoma cells were found to contain GD2 and/or GD3, whereas neuroblastoma cells expressed only GD2. Both antibodies induced high levels of ADCC and CDC to GD2/GD3-positive cells with human peripheral large granular lymphocytes (LGL) as effector cells and in the presence of human serum, respectively. A good correlation was obtained between the contents of disialogangliosides and the binding level of the antibodies; both melanoma and neuroblastoma cells with larger amounts of GD2/GD3 showed a higher level of antibody binding than did the cells with a smaller amount of GD2/GD3. Surprisingly, ADCC did not correlate well with the binding level of the antibodies. Thus, A1-425 showed stronger lytic activity than A1-267 in spite of the binding level of A1-425 being similar to or lower than that of A1-267 on the cell surfaces. Antigen-antibody complexes composed of GD2 and A1-425 showed higher binding levels to LGL than complexes of GD2 and A1-267. In contrast, free MAb molecules gave minimum binding to LGL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Developmental regulation of a gene coding for a low-molecular-weight heat shock protein during haustorium formation in the seedlings of a holoparasitic plant, Cuscuta japonica.

Dodder (Cuscuta japonica), a holoparasitic angiosperm, develops haustoria that are essential for parasitism. We have previously demonstrated that in Cuscuta seedlings, haustorial formation could be induced efficiently by cooperative effects of far-red light and tactile stimuli in the absence of any host plant [Tada et al. (1996) Plant Cell Physiol. 37: 1049]. In this study, we performed differential display and isolated several cDNAs that were expressed differentially during haustorium development in the seedlings. Sequence similarities identified one of them as a gene encoding a 17-kDa low-molecular-weight heat shock protein (CJHSP17). Northern blot analysis revealed that CJHSP17 mRNAs constitutively accumulated in the seedlings in the absence of environmental stress, and that the transcripts dramatically decreased to undetectable levels prior to emergence of haustoria upon irradiation with far-red light in the presence of tactile stimuli. When treated with either of the two stimuli, the CJHSP17 transcript levels did not decrease and there was no differentiation of haustoria. Moreover, irradiation of red light immediately after far-red light completely repressed both the decrease of mRNAs and the subsequent formation of haustoria. These observations suggest the involvement of down-regulation of CJHSP17 in haustorium development in Cuscuta seedlings.

Amino Acid Sequence↗
PubMed1988

Mutational analysis of the genome-linked protein VPg of poliovirus.

Using a mutagenesis cartridge (R. J. Kuhn, H. Tada, M. F. Ypma-Wong, J. J. Dunn, B. L. Semler, and E. Wimmer, Proc. Natl. Acad. Sci. USA 85:519-523, 1988), we have generated single and multiple amino acid replacement mutants, as well as a single amino acid insertion mutant in the genome-linked protein VPg of poliovirus. Moreover, we constructed three different 5-amino-acid insertion mutants that map close to the C terminus of 3A, a viral polypeptide whose coding sequence is adjacent to VPg. Transfection of HeLa cells with RNA synthesized in vitro was used to test the effect of the mutation on viral proliferation. Mutations were either lethal or nonlethal. A temperature-sensitive phenotype was not observed. The arginine at position 17 of VPg could not be exchanged with any other amino acid without loss of viability, whereas the lysine at position 20, an amino acid conserved among all known polioviruses, coxsackieviruses, and echoviruses, was replaceable with several neutral amino acids and even with glutamic acid. Replacement of poliovirus VPg with echovirus 9 VPg yielded viable virus with impaired growth properties. Our results suggest considerable flexibility in the amino acid sequence of a functional VPg. All insertions in polypeptide 3A proved to be lethal. In vitro translation of mutated viral RNAs gave patterns of proteolytic processing that in some cases was aberrant, even though the mutation was nonlethal.

Amino Acid Sequence↗
PubMed2000

Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway.

Gastrulation in the amphibian embryo is driven by cells of the mesoderm. One of the genes that confers mesodermal identity in Xenopus is Brachyury (Xbra), which is required for normal gastrulation movements and ultimately for posterior mesoderm and notochord differentiation in the development of all vertebrates. Xbra is a transcription activator, and interference with transcription activation leads to an inhibition of morphogenetic movements during gastrulation. To understand this process, we have screened for downstream target genes of Brachyury (Tada, M., Casey, E., Fairclough, L. and Smith, J. C. (1998) Development 125, 3997-4006). This approach has now allowed us to isolate Xwnt11, whose expression pattern is almost identical to that of Xbra at gastrula and early neurula stages. Activation of Xwnt11 is induced in an immediate-early fashion by Xbra and its expression in vivo is abolished by a dominant-interfering form of Xbra, Xbra-En(R). Overexpression of a dominant-negative form of Xwnt11, like overexpression of Xbra-En(R), inhibits convergent extension movements. This inhibition can be rescued by Dsh, a component of the Wnt signalling pathway and also by a truncated form of Dsh which cannot signal through the canonical Wnt pathway involving GSK-3 and (beta)-catenin. Together, our results suggest that the regulation of morphogenetic movements by Xwnt11 occurs through a pathway similar to that involved in planar polarity signalling in Drosophila.

Activins↗