Biomedical subjects

M Krinks

Publications and source records attributed to M Krinks.

10 recordsLinked to original sources

PubMed1997

Frzb-1, an antagonist of Wnt-1 and Wnt-8, does not block signaling by Wnts -3A, -5A, or -11.

Frzb-1 is a secreted factor that was recently shown to inhibit signaling by Wnts 1 and 8. Frzb-1 contains an amino terminal domain that is homologous to the Drosophila polarity gene frizzled; several other secreted proteins that share this feature have now been identified. These findings raise the question of whether or not Frzb-1 is a general inhibitor of signaling by all Wnts. We show that signaling of Wnts -3A, 5A, and 11 is not blocked by Frzb-1, demonstrating that Frzb-1 can discriminate both between the principle functional Wnt classes that have been proposed and within them.

Animals↗

PubMed1997

Frzb, a secreted protein expressed in the Spemann organizer, binds and inhibits Wnt-8.

We isolated a Xenopus homolog of Frzb, a newly described protein containing an amino-terminal Frizzled motif. It dorsalized Xenopus embryos and was expressed in the Spemann organizer during early gastrulation. Unlike Frizzled proteins, endogenous Frzb was soluble. Frzb was secretable and could act across cell boundaries. In several functional assays, Frzb antagonized Xwnt-8, a proposed ventralizing factor with an expression pattern complementary to that of Frzb. Furthermore, Frzb blocked induction of MyoD, an action reported recently for a dominant-negative Xwnt-8. Frzb coimmunoprecipitated with Wnt proteins, providing direct biochemical evidence for Frzb-Wnt interactions. These observations implicate Frzb in axial patterning and support the concept that Frzb binds and inactivates Xwnt-8 during gastrulation, preventing inappropriate ventral signaling in developing dorsal tissues.

Animals↗

PubMed1997

A novel Xenopus homologue of bone morphogenetic protein-7 (BMP-7).

We identified a Xenopus gene closely related to mammalian bone morphogenetic protein (BMP)-7 (also termed osteogenic protein-1 or OP-1). It resembles the mammalian gene in primary structure and expression pattern much more closely than does a previously described Xenopus homologue, originally termed XBMP-7 [Nishimatsu, Suzuki, Shoda, Murakami and Ueno (1992) Biochem. Biophys. Res. Commun. 186, 1487-1495]. The novel gene has therefore been designated XBMP-7 and the gene described earlier has been renamed XBMP-7R (M. Moos and N. Ueno, unpublished work). It has a broad distribution, primarily in the anterior and posterior ventral regions during gastrulation, subsequently becoming prominent at different stages in a wide variety of structures (eyes, neural structures, heart, pronephros, posterior ventral region and other structures), paralleling the distribution of XBMP-4 closely. However, its expression begins later than that of XBMP-4 during gastrulation. Lithium treatment of embryos concentrates the XBMP-7 expression in the expanded eye and heart structures. Ventral overexpression of XBMP-7 produces large protrusions that ultimately develop colouration characteristic of haemoglobin, which is confirmed by markedly expanded expression of alpha-globin. Dorsal overexpression suppresses dorsal anterior structures. Molecular analysis of animal caps overexpressing XBMP-7 reveals induction of markers associated with ventral and haematopoietic tissue, which is consistent with whole-embryo overexpression results. Globin induction by XBMP-7 can be blocked by a truncated BMP receptor previously shown to interrupt BMP-4 signalling, indicating XBMP-7 also interacts with this receptor. Our data support the concept that XBMP-7 may play a variety of roles during embryogenesis, and suggest a possible role in haematogenesis.

Amino Acid Sequence↗

PubMed1995

Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer.

We have identified a novel growth factor in Xenopus, which is most closely related to human Bone Morphogenetic Protein-3. Its expression peaks during gastrulation, most prominently in the Spemann organizer, and persists in the posterior neural floor plate and prechordal plate during neurulation. Injection of the corresponding mRNA into dorsal blastomeres results in dose-dependent suppression of dorsal and anterior structures, even in the presence of lithium chloride. Overexpression of the gene downregulates the dorsalizing factors noggin, goosecoid and follistatin, as well as the dorsal markers NCAM, muscle actin and MyoD; conversely, ventral markers are induced. We therefore designate this gene product Anti-Dorsalizing Morphogenetic Protein (ADMP). Though development of dorsoanterior structures is suppressed when exogenous ADMP is injected, the gene is induced by lithium chloride treatment or activin, both of which are known to produce the opposite effect. Thus, the expression of ADMP resembles that of several dorsalizing signals, but its product exerts dorsal-suppressing activity. This suggests that ADMP may moderate organizer-associated dorsalizing influences. These findings are also consistent with the recently advanced proposal of dorsally expressed inhibitory activin-like signals.

Amino Acid Sequence↗

PubMed1995

DNA sequencing from single phage plaques using solid-phase magnetic capture.

Many operations encountered in molecular cloning are labor-intensive and time-consuming. One case that is often troublesome is the subcloning of cDNA clones from lambda gt11 phage into plasmid vectors. In situations where several clones have been isolated, time could be saved by a means of assessing insert size and sequence unambiguously without subcloning, particularly where degenerate PCR or low-stringency hybridization approaches are taken to identify multiple members of a gene family. We describe a simple and reliable strategy for efficient sequencing of small amounts of lambda phage DNA, lysates or individual phage plaques. The strategy combines the advantages of universal lambda phage primers, rapid air thermal cycling, streptavidin magnetic bead capture of highly purified single-stranded templates and the unparalleled clarity of T7 DNA polymerase sequence. We routinely obtain 350-500 bases of unambiguous sequence from each reaction. It takes only hours from lifting phage plaques to finishing the sequencing reactions. The method provides an alternative to thermal cycle sequencing that has comparable sensitivity and affords sequence data of much higher clarity.

Bacteriophage lambda↗

PubMed1991

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy.

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities. The secondary structure of this protein in solution has been elucidated by a qualitative interpretation of nuclear Overhauser effects, hydrogen exchange data, and 3JHNH alpha coupling constants. A clear correlation between the 13C alpha chemical shift and secondary structure is found. The secondary structure in the two globular domains of Drosophila CaM in solution is essentially identical with that of the X-ray crystal structure of mammalian CaM [Babu, Y., Bugg, C. E., & Cook, W.J. (1988) J. Mol. Biol. 204, 191-204], which consists of two pairs of a "helix-loop-helix" motif in each globular domain. The existence of a short antiparallel beta-sheet between the two loops in each domain has been confirmed. The eight alpha-helix segments identified from the NMR data are located at Glu-6 to Phe-19, Thr-29 to Ser-38, Glu-45 to Glu-54, Phe-65 to Lys-77, Glu-82 to Asp-93, Ala-102 to Asn-111, Asp-118 to Glu-127, and Tyr-138 to Thr-146. Although the crystal structure has a long "central helix" from Phe-65 to Phe-92 that connects the two globular domains, NMR data indicate that residues Asp-78 to Ser-81 of this central helix adopt a nonhelical conformation with considerable flexibility.

Alanine↗

PubMed1991

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix.

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.

Amino Acid Sequence↗

PubMed1990

Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C. It is demonstrated that uniform 15N labeling permits the recording of sensitive three-dimensional (3D) NMR spectra that show far better resolution than their conventional two-dimensional analogs. Selective 15N labeling of amino acids can be used for identifying the type of amino acid, providing information that is essential for the analysis of the 3D spectra. Simultaneous selective labeling with both 15N and 13C can provide a number of unique backbone assignments from which sequential assignment can be continued.

Amino Acid Sequence↗

PubMed1990

An efficient NMR approach for obtaining sequence-specific resonance assignments of larger proteins based on multiple isotopic labeling.

By simultaneously incorporating in a protein 13C-carbonyl- and 15N-labeled amino acids with different levels of enrichment, characteristics asymmetric doublet-like patterns are observed for 15N nuclei that are directly adjacent to the 13C1-labeled residues, providing unambiguous identification of a large number of unique dipeptide fragments of the protein. Additional assignments and qualitative structural information can be obtained from such a selectively labeled protein by recording multiple bond correlation spectra. The procedure is demonstrated for the protein calmodulin, complexed with calcium.

Animals↗

PubMed1988

Identification, characterization, and functional correlation of calmodulin-dependent protein phosphatase in sperm.

Preliminary data demonstrated that the inhibition of reactivated sperm motility by calcium was correlated with inhibited protein phosphorylation. The inhibition of phosphorylation by Ca2+ was found to be catalyzed by the calmodulin-dependent protein phosphatase (calcineurin). Sperm from dog, pig, and sea urchin contain both the Ca2+-binding B subunit of the enzyme (Mr 15,000) and the calmodulin-binding A subunit with an Mr of 63,000. The sperm A subunit is slightly higher in Mr than reported for other tissues. Inhibition of endogenous calmodulin-dependent protein phosphatase activity with a monospecific antibody revealed the presence of 14 phosphoprotein substrates in sperm for this enzyme. The enzyme was localized to both the flagellum and the postacrosomal region of the sperm head. The flagellar phosphatase activity was quantitatively extracted with 0.6 M KCl from isolated flagella from dog, pig, and sea urchin sperm. All salt-extractable phosphatase activity was inhibited with antibodies against the authentic enzyme. Preincubation of sperm models with the purified phosphatase stimulated curvolinear velocity and lateral head amplitude (important components of hyperactivated swimming patterns) and inhibited beat cross frequency suggesting a role for this enzyme in axonemal function. Our results suggest that calmodulin-dependent protein phosphatase plays a major role in the calcium-dependent regulation of flagellar motility.

Animals↗