Hypoxia up-regulates triosephosphate isomerase expression via a HIF-dependent pathway.

The glycolytic enzyme triosephosphate isomerase (TPI) catalyses the reversible conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We report here that the expression of TPI at both the mRNA and protein levels is increased by hypoxia in vivo and in vitro. The temporal pattern of hypoxic TPI induction is very similar to that of genes triggered by the hypoxia-inducible transcription factor (HIF) and is mimicked characteristically by cobalt and by deferoxamine, but is absent in cells with a defective aryl hydrocarbon receptor nuclear translocator (ARNT, here HIF-1beta) and in cells lacking HIF-1alpha protein. We conclude from these findings that the expression of TPI is regulated via the HIF pathway and thus belongs to the family of classic oxygen-regulated genes. The physiological meaning of an increased expression of TPI in hypoxygenated tissues is probably to increase the flow of triosephosphates through the glycolytic cascade thus leading to an increase of anaerobic energy generation.

Hydra and Niccolo Paganini (1782-1840)--two peas in a pod? The molecular basis of extracellular matrix structure in the invertebrate, Hydra.

The body wall of Hydra is organized as an epithelial bilayer with an intervening extracellular matrix (ECM). Molecular and biochemical analyses of Hydra ECM have established that it contains components similar to those seen in more complicated vertebrates such as human. In terms of biophysical parameters, Hydra ECM is highly flexible; a property that facilitates continuous movements along the organism's longitudinal and radial axis. A more rigid ECM, as in vertebrates, would not be compatible with this degree of movement. The flexible nature of Hydra ECM can now be explained in part by the unique structure of the organism's collagens. Interestingly, some aspects of the structural features of Hydra collagens mimic what is seen in Ehlers-Danlos syndrome, an inherited condition in humans that results in an abnormally flexible ECM that can be debilitating in extreme cases. This review will focus on structure-function relationships of the ECM of Hydra.

Silica-precipitating peptides from diatoms. The chemical structure of silaffin-A from Cylindrotheca fusiformis.

Two silica-precipitating peptides, silaffin-1A(1) and-1A(2), both encoded by the sil1 gene from the diatom Cylindrotheca fusiformis, were extracted from cell walls and purified to homogeneity. The chemical structures were determined by protein chemical methods combined with mass spectrometry. Silaffin-1A(1) and -1A(2) consist of 15 and 18 amino acid residues, respectively. Each peptide contains a total of four lysine residues, which are all found to be post-translationally modified. In silaffin-1A(2) the lysine residues are clustered in two pairs in which the epsilon-amino group of the first residue is linked to a linear polyamine consisting of 5 to 11 N-methylated propylamine units, whereas the second lysine is converted to epsilon-N,N-dimethyllysine. Silaffin-1A(1) contains only a single lysine pair exhibiting the same structural features. One of the two remaining lysine residues was identified as epsilon-N,N,N-trimethyl-delta-hydroxylysine, a lysine derivative containing a quaternary ammonium group. The fourth lysine residue again is linked to a long-chain polyamine. Silaffin-1A(1) is the first peptide shown to contain epsilon-N,N,N-trimethyl-delta-hydroxylysine. In vitro, both peptides precipitate silica nanospheres within seconds when added to a monosilicic acid solution.

Laminin isoforms 8 and 10 are primary components of the subendothelial basement membrane promoting interaction with neoplastic lymphocytes.

To determine whether subendothelial laminins (LNs) could be implicated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluorescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758-762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffinity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid sequencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached preferentially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.

Species-specific polyamines from diatoms control silica morphology.

Biomineralizing organisms use organic molecules to generate species-specific mineral patterns. Here, we describe the chemical structure of long-chain polyamines (up to 20 repeated units), which represent the main organic constituent of diatom biosilica. These substances are the longest polyamine chains found in nature and induce rapid silica precipitation from a silicic acid solution. Each diatom is equipped with a species-specific set of polyamines and silica-precipitating proteins, which are termed silaffins. Different morphologies of precipitating silica can be generated by polyamines of different chain lengths as well as by a synergistic action of long-chain polyamines and silaffins.

Molecular, biochemical and functional analysis of a novel and developmentally important fibrillar collagen (Hcol-I) in hydra.

The body wall of hydra (a member of the phylum Cnidaria) is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Previous studies have established that cell-ECM interactions are important for morphogenesis and cell differentiation in this simple metazoan. The ECM of hydra is particularly interesting because it represents a primordial form of matrix. Despite progress in our understanding of hydra ECM, we still know little about the nature of hydra collagens. In the current study we provide a molecular, biochemical and functional analysis of a hydra fibrillar collagen that has similarity to vertebrate type I and type II collagens. This fibrillar collagen has been named hydra collagen-I (Hcol-I) because of its structure and because it is the first ECM collagen to be identified in hydra. It represents a novel member of the collagen family. Similar to vertebrate type I and II collagens, Hcol-I contains an N-terminal propeptide-like domain, a triple helical domain containing typical Gly-X-Y repeats and a C-terminal propeptide domain. The overall identity to vertebrate fibrillar collagens is about 30%, while the identity of the C-terminal propeptide domain is 50%. Because the N-terminal propeptide domain is retained after post-translational processing, Hcol-I does not form thick fibers as seen in vertebrates. This was confirmed using transmission electron microscopy to study rotary shadow images of purified Hcol-I. In addition, absence of crucial lysine residues and an overall reduction in proline content, results in reduced crosslinking of fibrils and increased flexibility of the molecule, respectively. These structural changes in Hcol-I help to explain the flexible properties of hydra ECM. Immunocytochemical studies indicate that Hcol-I forms the 10 nm fibrils that comprise the majority of molecules in the central fibrous zone of hydra ECM. The central fibrous zone resides between the two subepithelial zones where hydra laminin is localized. While previous studies have shown that basal lamina components like laminin are expressed by the endoderm, in situ hybridisation studies show that Hcol-I mRNA expression is restricted to the ectoderm. Hcol-I expression is upregulated during head regeneration, and antisense studies using thio-oligonucleotides demonstrated that blocking the translation of Hcol-I leads to a reversible inhibition of head morphogenesis during this regenerative process. Taken in total, the data presented in this study indicate that Hcol-I is required for morphogensis in hydra and represents a novel fibrillar collagen whose structural characteristics help to explain the unique biophysical properties of hydra ECM. Interestingly, the structure of Hcol-I mimics what is seen in Ehlers-Danlos syndrome type VII in humans; an inherited pathological condition that leads to joint and skin abnormalities. Hcol-I therefore illustrates an adaptive trait in which the normal physiological situation in hydra translates into a pathological condition in humans.

Characterization of hydra type IV collagen. Type IV collagen is essential for head regeneration and its expression is up-regulated upon exposure to glucose.

Hydra vulgaris mesoglea is a primitive basement membrane that also exhibits some features of an interstitial matrix. We have characterized cDNAs that encode the full-length hydra alpha1(IV) chain. The 5169-base pair transcript encodes a protein of 1723 amino acids, including an interrupted 1455-residue collagenous domain and a 228-residue C-terminal noncollagenous domain. N-terminal sequence analyses of collagen IV peptides suggest the molecule is homotrimeric. Denatured hydra type IV collagen protein occurs as dimers and higher order aggregates held together by nonreducible cross-links. Hydra collagen IV exhibits no functional evidence for the presence of a 7 S domain. Type IV collagen is expressed by the ectoderm along the entire longitudinal axis of the animal but is most intense at the base of the tentacles at the site of battery cell transdifferentiation. Antisense studies show that inhibition of collagen IV translation causes a blockage in head regeneration, indicating its importance in normal hydra development. Exposure of adult hydra to 15 mm glucose resulted in up-regulation of type IV collagen mRNA levels within 48 h and significant thickening of the mesoglea within 14 days, suggesting that basement membrane thickening seen in diabetes may be, in evolutionary terms, an ancient glucose-mediated response.

Sulfite:Cytochrome c oxidoreductase from Thiobacillus novellus. Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family.

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC ). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alphabeta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c(552) subunit (midpoint redox potential, E(m8.0) = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K(m) values for sulfite and cytochrome c(550) were determined to be 27 and 4 micrometer, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Polycationic peptides from diatom biosilica that direct silica nanosphere formation.

Diatom cell walls are regarded as a paradigm for controlled production of nanostructured silica, but the mechanisms allowing biosilicification to proceed at ambient temperature at high rates have remained enigmatic. A set of polycationic peptides (called silaffins) isolated from diatom cell walls were shown to generate networks of silica nanospheres within seconds when added to a solution of silicic acid. Silaffins contain covalently modified lysine-lysine elements. The first lysine bears a polyamine consisting of 6 to 11 repeats of the N-methyl-propylamine unit. The second lysine was identified as epsilon-N,N-dimethyl-lysine. These modifications drastically influence the silica-precipitating activity of silaffins.

EMILIN, a component of the elastic fiber and a new member of the C1q/tumor necrosis factor superfamily of proteins.

EMILIN (elastin microfibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil alpha helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of beta-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative.

Analysis of heparin, alpha-dystroglycan and sulfatide binding to the G domain of the laminin alpha1 chain by site-directed mutagenesis.

The 395-residue proteolytic fragment E3, which comprises the two most C-terminal LG modules of the mouse laminin alpha1 chain, was previously shown to contain major binding sites for heparin, alpha-dystroglycan and sulfatides. The same fragment (alpha1LG4-5) and its individual alpha1LG4 and alpha1LG5 modules have now been obtained by recombinant production in mammalian cells. These fragments were apparently folded into a native form, as shown by circular dichroism, electron microscopy and immunological assays. Fragment alpha1LG4-5 bound about five- to tenfold better to heparin, alpha-dystroglycan and sulfatides than E3. These binding activities could be exclusively localized to the alpha1LG4 module. Side-chain modifications and proteolysis demonstrated that Lys and Arg residues in the C-terminal region of alpha1LG4 are essential for heparin binding. This was confirmed by 14 single to triple point mutations, which identified three non-contiguous basic regions (positions 2766-2770, 2791-2793, 2819-2820) as contributing to both heparin and sulfatide binding. Two of these regions were also recognized by monoclonal antibodies which have previously been shown to inhibit heparin binding. The same three regions and a few additional basic residues also make major contributions to the binding of the cellular receptor alpha-dystroglycan, indicating a larger binding epitope. The data are also consistent with previous findings that heparin competes for alpha-dystroglycan binding.

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium.

The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N-glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser-, in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N-glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.

Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli.

Proteins which are synthesized with a signal peptide containing a 'double-arginine' motif may be translocated across the bacterial cytoplasmic membrane by a mechanism that is different from the known Sec and signal recognition particle pathways. The function of the double-arginine motif as a determinant for this novel pathway was studied by expressions of gene constructs coding for the high potential iron-sulfur protein (HiPIP) from Chromatium vinosum D in Escherichia coli. When the protein was produced with its original double-arginine motif-containing signal peptide, it was in part translocated into the periplasm and thereby processed, as shown by immunoblots after cell fractionation and N-terminal sequencing of purified HiPIP. Processing was not inhibited significantly by 3 mM sodium azide, indicating that translocation of HiPIP occurs by a SecA-independent pathway. Translocation of HiPIP could be altered to the SecA-dependent mode when its signal peptide was substituted by that of PelB from Erwinia carotovora. When the HiPIP double-arginine motif (SRRDAVK) was introduced into the corresponding position of the PelB signal peptide, the transport pathway remained SecA-dependent. This indicates that additional determinants are required for translocation by the Sec-independent pathway.

Molecular characterization of cyanophycin synthetase, the enzyme catalyzing the biosynthesis of the cyanobacterial reserve material multi-L-arginyl-poly-L-aspartate (cyanophycin).

Cyanophycin (multi-L-arginyl-poly-L-aspartate), a water-insoluble reserve polymer of cyanobacteria, is a product of nonribosomal peptide synthesis. The purification of cyanophycin synthetase of the cyanobacterium Anabaena variabilis is described. In sodium dodecylsulfate/polyacrylamide gel electrophoresis, the enzyme preparation shows one band with an apparent molecular mass of 100 kDa. The native enzyme has an apparent molecular mass of approximately 230 kDa, as determined by size-exclusion chromatography, suggesting that the active form is a homodimer. During catalysis, ATP is converted to ADP. The gene coding for cyanophycin synthetase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The C-terminal 60% of the deduced amino acid sequence of cyanophycin synthetase show sequence similarity to enzymes of the superfamily of ligases involved in the biosynthesis of murein and of folyl-poly(gamma-glutamate). Cells of Escherichia coli harbouring the gene on a plasmid express active synthetase and accumulate cyanophycin-like material. The results prove that a single enzyme catalyzes the de novo synthesis of cyanophycin.

Developmental regulation of the laminin alpha5 chain suggests a role in epithelial and endothelial cell maturation.

We have previously shown that mouse and bovine endothelial cells express a novel 400-kDa laminin alpha chain complexed to beta1 and gamma1 laminin chains. We describe here purification of this laminin isoform from the conditioned medium of a mouse peripheral lymph node endothelial cell line, SVEC. The laminin alpha chain was isolated from the laminin complex, subjected to Edman digestion, and the amino acid sequences of the resulting peptides were determined. Amino acid sequence revealed 100% identity to the predicted amino acid sequence of the recently reported laminin alpha5 gene. A monoclonal antibody to the laminin alpha5 chain was raised (4G6), allowing investigation of its distribution in embryonic, newborn, and mature mouse tissues. The laminin alpha5 chain was expressed mainly by epithelial, endothelial, and myogenic cells: In both embryonic and mature tissues the laminin alpha5 chain was strongly expressed by epithelial cells, the bronchi of the lungs and the developing kidney tubules being the sites of strongest expression. However, laminin alpha5 was not associated with early stages of epithelial cell development, but rather with epithelial cell maturation. Widespread expression of laminin alpha5 in endothelial cells was apparent only in tissues of mature mice, its appearance correlating approximately with sexual maturity. During embryogenesis and in newborn tissues, laminin alpha5 occurred in basement membranes of larger blood vessels only, excluding a role in angiogenic processes. Smooth muscle and skeletal muscle cells were the only other cell types which showed considerable laminin alpha5 expression, with skeletal muscle exhibiting a developmentally regulated pattern of expression: The laminin alpha5 chain occurred in skeletal muscle fiber basement membranes early in embryogenesis (E13-E15) but decreased with development, remaining strongly expressed only at the neuromuscular junction. The data show that laminin alpha5 expression is associated with epithelial and endothelial cell maturation, implicating a role for this laminin chain in the maintenance of differentiated epithelial and endothelial cell phenotype.

Cloning of the mouse laminin alpha 4 cDNA. Expression in a subset of endothelium.

Endothelial cells express a 400-kDa or 240-kDa laminin alpha chain, depending on their tissue of origin or physiological state [1, 2]. Using differential display and subsequent screening of a mouse endothelial cell cDNA library we here identify the gene coding for the 240-kDa laminin chain as the laminin alpha 4 gene. The complete mouse laminin alpha 4 cDNA sequence is reported and compared with other laminin alpha chains. In situ hybridization of embryonic and new born mouse tissues revealed expression of laminin alpha 4 mRNA in a subset of endothelium, in particular aortic endothelium, endocardium and endothelium of blood vessels in the skin and in the brain. Strong laminin alpha 4 expression by aortic endothelia was confirmed by data obtained from cultured bovine aortic endothelial cells (BAEC). Isolation of laminin from BAEC conditioned medium revealed a Y-shaped molecule in rotary shadowing. Subsequent sequencing of BAEC laminin resulted in laminin alpha 4, beta 1 and gamma 1 amino acid sequences, confirming that laminin alpha 4 is one of the major laminin alpha chains expressed by aortic endothelium not only in the mouse. In addition, strong laminin alpha 4 mRNA expression occurred in peripheral nerves, cardiac muscle, fat, the dermis of the skin and lung stroma of mouse tissues. The data demonstrate a cytokine and progesterone-regulated differential expression of laminin alpha 4 mRNA in mouse endothelium, suggesting a distinct functional role for this laminin chain in endothelium.

HNK-1 carbohydrate-mediated cell adhesion to laminin-1 is different from heparin-mediated and sulfatide-mediated cell adhesion.

The sulfated HNK-1 carbohydrate present on glycolipids and on several neural recognition molecules has been shown to mediate the adhesion of murine small cerebellar neurons and astrocytes to the extracellular matrix molecule laminin-1. In this study, we characterized the binding of the HNK-1 carbohydrate to laminin-1 extracted from the Engelbreth-Holm-Swarm (EHS) sarcoma and distinguished it unequivocally from binding sites for other sulfated carbohydrates. Electron microscopic analysis of rotary shadowed complexes of laminin-1 and a HNK-1 neoglycoprotein revealed a major binding site on the G domain that comprises the C-terminal globule of the laminin alpha1 chain. The HNK-1 carbohydrate also interacted with placental laminin isoforms containing an alpha chain variant. It bound to the proteolytic laminin-1 fragment E8 comprising the domains G1-G3, but not to fragment E3 that carries the major heparin-binding site on domains G4-G5. No binding was observed to the short arm containing fragments E1XNd or P1. Binding studies with native or denatured laminin E8 fragments and proteolytic or recombinant fragments of the G domain localized the HNK-1 carbohydrate binding site to domain G2. The binding could be clearly distinguished from binding sites for other sulfated carbohydrates such as heparin and sulfatides. Further, the binding could not be abolished by reduction and alkylation or by urea treatment of laminin-1 and was independent of the native conformation of laminin-1 and of Ca2+. The G2 domain is also involved in the adhesion of HNK-1 carbohydrate expressing early postnatal cerebellar neurons and is different from heparin- and sulfatide-mediated cell adhesion to laminin-1. HNK-1 carbohydrate-mediated cell adhesion appears, however, to be dependent on the native conformation of laminin-1 indicating a more complex cellular recognition process.

Fritz: a secreted frizzled-related protein that inhibits Wnt activity.

Signaling molecules of the Wnt gene family are involved in the regulation of dorso-ventral, segmental and tissue polarity in Xenopus and Drosophila embryos. Members of the frizzled gene family, such as Drosophila frizzled-2 and rat frizzled-1, have been shown encode Wnt binding activity and to engage intracellular signal transduction molecules known to be part of the Wnt signaling pathway. Here we describe the cloning and characterization of Fritz, a mouse (mfiz) and human (hfiz) gene which codes for a secreted protein that is structurally related to the extracellular portion of the frizzled genes from Drosophila and vertebrates. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. We propose a model in which Fritz can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites.

Biochemical characterization and microsequencing of a 205-kDa synovial protein stimulatory for T cells and reactive with rheumatoid factor containing sera.

Synovial fluid (SF) was found to possess stimulatory capacity for the proliferation of T cell clones derived from patients with rheumatoid arthritis (RA) when cultured together with IL-2. Using chromatography technique and gel electrophoresis, a synovial fluid protein with an apparent m.w. of 205 kDa (p205) was isolated that demonstrated a bioactivity analogous to that obtained with native synovial fluid. After electroelution, p205 dissociated into 70-kDa fragment(s). Upon IEF, it appeared as a single band with an isoelectric point of 6.5, suggesting a noncovalently bound trimer complex. Amino acid sequences of the whole protein and of tryptic peptides were determined by N terminal sequencing. The N terminal amino acid sequence of the 70-kDa fragment and of the tryptic peptides showed no identity to recently described protein sequences. One peptide matched, in 11 amino acid residues, with the human IgG1-4 constant heavy chain and rheumatoid factor (RF) binding region. The p205 induced the proliferation of peripheral blood T cells and long term T cell cultures that had been raised by alternate stimulation with IL-2 and p205. In a similar approach, synovial lining cells were shown to release a protein with biochemical characteristics similar to the synovial fluid-derived p205. Western blot analysis revealed the binding of RF-containing sera to p205, which was diminished by absorption with an RF reagent. These observations suggest that p205 is expressed by synovial cells and may be a target for T and B cells in RA.

Stable photobleaching of P840 in Chlorobium reaction center preparations: presence of the 42-kDa bacteriochlorophyll a protein and a 17-kDa polypeptide.

Simple procedures for the anaerobic preparation of photoactive and stable P840 reaction centers from Chlorobium tepidum and Chlorobium limicola in good yield are presented and quantitated. The subunit composition was tested by cosedimentation in sucrose density gradients. For C. limicola, it minimally comprises four subunits: the P840 reaction center protein PscA, the BChla antenna protein FMO, the FeS protein PscB with centers A and B, and a positively charged 17-kDa protein denoted PscD. The preparation from Chlorobium tepidum additionally contained PscC, a cytochrome c-551. The BChla absorption peak of the purified complexes was at 810 nm, with a shoulder at 835 nm. The ratio of the shoulder to the peak was 0.25, which corresponds to 1 reaction center per 70 BChla molecules if a uniform extinction coefficient of BChla is assumed. However, bleaching at 610 nm in continuous light corresponded up to 1 photoactive reaction center per 50 BChla molecules. Therefore, either the extinction coefficient of BChla in the reaction center is overestimated or the one for photobleaching is underestimated. In any case, the major portion of the reaction center was photoactive in the preparations. A P840 reaction center subcomplex, lacking PscD and deficient in FMO and PscB, but retaining the cytochrome c subunit, was obtained as a side product. It was photoinactive and had an absorption peak at 814 nm and a 835/814 absorbance ratio of 0.42. FMO and PscB show the tendency to form a complementary subcomplex. FMO and PscD are apparently required to stabilize the photoactive reaction center, while the cytochrome c subunit is not.