Olfactory and lens placode formation is controlled by the hedgehog-interacting protein (Xhip) in Xenopus.

The integration of multiple signaling pathways is a key issue in several aspects of embryonic development. In this context, extracellular inhibitors of secreted growth factors play an important role, which is to antagonize specifically the activity of the corresponding signaling molecule. We provide evidence that the Hedgehog-interacting protein (Hip) from Xenopus, previously described as a Hedgehog-specific antagonist in the mouse, interferes with Wnt-8 and eFgf/Fgf-8 signaling pathways as well.

Microtubule-associated protein 1S, a short and ubiquitously expressed member of the microtubule-associated protein 1 family.

The related high molecular mass microtubule-associated proteins (MAPs) MAP1A and MAP1B are predominantly expressed in the nervous system and are involved in axon guidance and synaptic function. MAP1B is implicated in fragile X mental retardation, giant axonal neuropathy, and ataxia type 1. We report the functional characterization of a novel member of the microtubule-associated protein 1 family, which we termed MAP1S (corresponding to sequence data bank entries for VCY2IP1 and C19ORF5).

Nuclear localization destabilizes the stress-regulated transcription factor Msn2.

The transcriptional program of yeast cells undergoes dramatic changes during the shift from fermentative growth to respiratory growth. A large part of this response is mediated by the stress responsive transcription factor Msn2. During glucose exhaustion, Msn2 is activated and concentrated in the nucleus.

High-pressure cryoimmobilization of murine skin reveals novel structural features and prevents extraction artifacts.

Cryoimmobilization by high-pressure freezing (HPF) and subsequent freeze substitution has been proven as an effective method to preserve tissues. Here, we demonstrate for the first time that a comprehensive morphological and ultrastructural preservation of mouse skin throughout all its layers can be achieved in this way. Using conditions limiting tissue-extraction during freeze substitution, we could prevent the massive interdigitation of cell membranes, the loss of tubular structures of the Golgi complex, the aggregation of keratin to electron-dense bundles, the formation of round-shaped keratohyalin aggregates, the dispersion of locally organized ribosomes, the excessive aggregation of material at hemidesmosomal plaques, the massive extraction of material from the basement membrane and the adjacent dermal region, and the dissociation of components of the dermal matrix.

Plectin-RACK1 (receptor for activated C kinase 1) scaffolding: a novel mechanism to regulate protein kinase C activity.

Agonist-induced translocation of protein kinase C (PKC) isozymes is mediated by receptors for the activated form of the kinase, shuttling it from one intracellular site to another and enhancing its catalytic activity. It is however unknown whether the receptors themselves are anchored to certain intracellular structures prior to their engagement with PKC. We show here sequestering of receptor for activated C kinase 1 (RACK1) to the cytoskeleton through the cytoskeletal linker protein plectin during the initial stages of cell adhesion.

Differences in the involvement of prostanoids from Kupffer cells in the mediation of anaphylatoxin C5a-, zymosan-, and lipopolysaccharide-dependent hepatic glucose output and flow reduction.

Various inflammatory stimuli such as anaphylatoxin C5a, zymosan, and lipopolysaccharides (LPSs) have been reported both to enhance glucose output in the perfused rat liver and to induce prostanoid (ie, prostaglandin and thromboxane) release from Kupffer cells, the resident liver macrophages. Because prostanoids can enhance glucose output from hepatocytes, it was the aim of this study to compare the possible roles of prostanoids released after C5a, zymosan, and LPS in the mediation of hepatic glucose output. In perfused livers both C5a and zymosan immediately enhanced glucose output, reduced flow, and induced prostanoid overflow into the hepatic vein, but with different quantities and kinetics.

High-pressure freezing of epithelial cells on sapphire coverslips.

Rapid freezing of cell monolayers at ambient pressure is limited regarding the thickness of ice crystal damage-free freezing. The specific freezing conditions of the cells under investigation are decisive for the success of such methods. Improved reproducibility of results could be expected by cryoimmobilization at high pressure because this achieves a greater thickness of adequate freezing.

Plectin 5'-transcript diversity: short alternative sequences determine stability of gene products, initiation of translation and subcellular localization of isoforms.

Plectin is a large cytoskeletal linker protein expressed as several different isoforms from a highly complex gene. This transcript diversity is mainly caused by short 5'-sequences contained in alternative first exons. To elucidate the influence of these sequence differences and to determine potential differential functionality of the resulting protein forms, we conducted a systematic investigation of plectin isoforms on transcript and protein levels.

A restrictive role for Hedgehog signalling during otic specification in Xenopus.

Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches.

Epiplakin gene analysis in mouse reveals a single exon encoding a 725-kDa protein with expression restricted to epithelial tissues.

Based on cDNA cloning and sequencing, human epiplakin has been classified as a member of the plakin protein family of cytolinkers. We report here the characterization of the mouse epiplakin gene locus and the isolation of full-length mouse epiplakin cDNA using BAC vectors. We found that the protein is encoded by a single remarkably large exon (>20 kb) that consists of a series of 0.

Cryofixation of epithelial cells grown on sapphire coverslips by impact freezing.

Rapid cryofixation of cells cultured on coverslips without the use of chemical fixatives has proved advantageous for the immunolocalization of antigens by electron microscopy. Here, we demonstrate the application of sapphire-attached tissue culture cells (PtK2 epithelial cells and mouse myoblasts) to metal-mirror impact freezing. The potential of the Leica EM-CPC cryoworkstation for routine freezing and for safe transfer of the cryofrozen samples into a sapphire disc magazine for freeze-substitution (SD-FS unit) has been exploited.

Plectin-isoform-specific rescue of hemidesmosomal defects in plectin (-/-) keratinocytes.

The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes.

Direct binding of plectin to Fer kinase and negative regulation of its catalytic activity.

Plectin is a cyoskeletal linker protein that protects tissues against mechanical stress. We report here that the N-terminal domain of the nonreceptor tyrosine kinase Fer interacts with N-terminal sequences of plectin. Recombinant protein encoded by exon 12-24 of rat plectin bound directly to amino acid 1-329 of murine Fer.

FIP-2, an IkappaB-kinase-gamma-related protein, is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation.

Using an antiserum directed against marginal band associated proteins of chicken erythrocytes we isolated clones encoding the chicken homolog of 14.7K-interacting protein 2 (FIP-2), a protein potentially involved in tumor necrosis factor-alpha/nuclear factor-kappaB signaling, from a chicken erythroblast cDNA library. We found that chicken FIP-2 was expressed in a variety of tissues and cell types, but unlike its human counterpart, alternative splicing does not appear to take place.

Gli-type zinc finger proteins as bipotential transducers of Hedgehog signaling.

Secreted proteins of the Hedgehog (Hh) family direct the development of diverse organs and tissues of vertebrates and invertebrates. Gli-type zinc finger proteins function as transcriptional mediators of the Hh signaling cascade and were implicated both in the activation and repression of Hh target genes. The differential activity of Gli-type zinc finger proteins is regulated on the level of proteolytic processing and subcellular localization as a complex concert of Hh-responsive, intracellular determinants.

XSPR-1 and XSPR-2, novel Sp1 related zinc finger containing genes, are dynamically expressed during Xenopus embryogenesis.

Proteins related to the human transcription factor Sp1 are characterized by the presence of a highly conserved zinc finger domain consisting of three C2H2 type zinc fingers. Here we describe two Xenopus laevis cDNAs, which encode novel Sp1-related C2H2 type zinc finger transcription factors named XSPR-1 and XSPR-2. Structurally, XSPR-1 and XSPR-2 are closely related to the murine Sp5, which interacts genetically with Brachyury (Dev.

Microtubule-associated protein 1A (MAP1A) and MAP1B: light chains determine distinct functional properties.

The microtubule-associated proteins 1A (MAP1A) and 1B (MAP1B) are distantly related protein complexes consisting of heavy and light chains and are thought to play a role in regulating the neuronal cytoskeleton, MAP1B during neuritogenesis and MAP1A in mature neurons. To elucidate functional differences between MAP1B and MAP1A and to determine the role of the light chain in the MAP1A protein complex, we chose to investigate the functional properties of the light chain of MAP1A (LC2) and compare them with the light chain of MAP1B (LC1). We found that LC2 binds to microtubules in vivo and in vitro and induces rapid polymerization of tubulin.

Plectin repeats and modules: strategic cysteines and their presumed impact on cytolinker functions.

Plectin, a member of the cytolinkers protein family, plays a crucial role in cells as a stabilizing element of cells against mechanical stress. Its absence results in muscular dystrophy, skin blistering, and signs of neuropathy. The C-terminal domain of plectin contains several highly homologous repeat domains that also occur in other cytolinkers.

Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast.

Rck2, a yeast Ser/Thr protein kinase homologous to mammalian calmodulin kinases, requires phosphorylation for activation. We provide evidence that in budding yeast, this step can be executed by the osmostress-activated mitogen-activated protein kinase Hog1. Rck2 phosphorylation was transiently increased during osmostress or in mutants with a hyperactive high osmolarity glycerol (HOG) pathway.

Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p.

The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3). Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated. They overlapped in the region of amino acids 318-550.

Xpitx3: a member of the Rieg/Pitx gene family expressed during pituitary and lens formation in Xenopus laevis.

Xpitx3 is the Xenopus homologue of the mouse Pitx3 gene and belongs to the family of RIEG/PITX homeobox genes. Here, we report on the embryonic expression of Xpitx3. It is transcribed in the presumptive pituitary already at the open neural tube stage.

The molecular peculiarities of catalase-peroxidases.

In developing ideas of how protein structure modifies haem reactivity, the activity of Class I of the plant peroxidase superfamily (including cytochrome c peroxidase, ascorbate peroxidase and catalase-peroxidases (KatGs)) is an exciting field of research. Despite striking sequence homologies, there are dramatic differences in catalytic activity and substrate specificity with KatGs being the only member with substantial catalase activity. Based on multiple sequence alignment performed for Class I peroxidases, we present a hypothesis for the pronounced catalase activity of KatGs.

Xnkx-2.1: a homeobox gene expressed during early forebrain, lung and thyroid development in Xenopus laevis.

Nkx-2.1 is a member of the vertebrate Nkx family of homeobox genes; it was originally identified as a tissue-specific regulator of thyroglobulin and thyroperoxidase gene transcription. Here we report on the embryonic expression of Xnkx-2.

Molecular cloning and expression analysis of the Hedgehog receptors XPtc1 and XSmo in Xenopus laevis.

Inductive signaling mediated by secreted factors of the Hedgehog (Hh) gene family regulates cellular proliferation and differentiation in many embryonic tissues. Two transmembrane proteins associated in a complex, Patched (Ptc) and Smoothened (Smo), are indispensable for the reception of Hh signals (Cell 86 (1996) 221; Nature 382 (1996) 547; Nature 384 (1996) 176; Nature 384 (1996) 129). Here, we report on the identification of Ptc and Smo homologues from Xenopus and analyze their spatio-temporal expression during embryogenesis.