Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.     

Cys-140 is critical for metabotropic glutamate receptor-1 dimerization

Metabotropic glutamate receptor 1 (mGluR1) expresses at the cell surface as disulfide-linked dimers and can be reduced to monomers with sulfhydryl reagents. To identify the dimerization domain, we transiently expressed in HEK-293 cells a truncated version of mGluR1 (RhodC-R1) devoid of the extracellular domain (ECD). RhodC-R1 was a monomer in the absence or presence of the reducing agents, suggesting that dimerization occurs via the ECD. To identify cysteine residues involved in dimerization within the ECD, cysteine to serine point mutations were made at three cysteines within the amino-terminal half of the ECD. A mutation at positions Cys-67, Cys-109, and Cys-140 all resulted in significant amounts of monomers in the absence of reducing agents. The monomeric C67S and C109S mutants were not properly glycosylated, failed to reach the cell surface, and showed no glutamate response, indicating that these mutant receptors were improperly folded and/or processed and thus retained intracellularly. In contrast, the monomeric C140S mutant was properly glycosylated, processed, and expressed at the cell surface. Phosphoinositide hydrolysis assay showed that the glutamate response of the C140S mutant receptor was similar to the wild type receptor. Substitution of a cysteine for Ser-129, Lys-134, Asp-143, and Thr-146 on the C140S mutant background restored receptor dimerization. Taken together, the results suggest that Cys-140 contributes to intermolecular disulfide-linked dimerization of mGluR1.     

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.     

Structural requirements for dimerization,glycosylation, secretion,and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosyaated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.     

Rat procathepsin B. Proteolytic processing to the mature form in vitro.

Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.     

Human bradykinin B2 receptor sialylation and N-glycosylation participate with disulfide bonding in surface receptor dimerization

G-Protein-coupled receptors (GPCRs) act on the cell surface where they recognize and convert external stimuli to modulate cellular activity and are regulated by agonist and various partner molecules. We here studied the cell surface post-translationally modified forms of a GPCR, the human bradykinin B2 receptor. This was by means of detailed molecular analysis of the cell surface forms of N-glycosylation site mutant and wild-type receptors that were treated with glycosidases, neuraminidase, and/or the reducing agent dithiothreitol or not treated before Western blotting. We found that the receptor undergoes similar glycosylation processes and similar cell surface organization in CHO-K1 and HEK 293 cells, used for stable and transient receptor expression, respectively. The receptor is present as dimers and monomers on the cell surface. The dimers result from heterologous association of differently glycosylated mature receptor molecules. Importantly, receptor sialylation and N-glycosylation participate with disulfide bonding in the stabilization of the cell surface human B2 receptor dimers.     

Mechanism of origin activation by monomers of R6K-encoded pi protein

One recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific Rep proteins that bind to DNA sequences called iterons. For plasmid R6K, this process involves a complex interplay between monomers and dimers of the Rep protein, pi, with seven tandem iterons of gamma ori. Remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. Dimers, the predominant form in the cell, inhibit replication, while monomers facilitate open complex formation and activate the ori. Here, we investigate a mechanism by which pi monomers out-compete pi dimers for iteron binding, and in so doing activate the ori. With an in vivo plasmid incompatibility assay, we find that pi monomers bind cooperatively to two adjacent iterons. Cooperative binding is eliminated by insertion of a half-helical turn between two iterons but is diminished only slightly by insertion of a full helical turn between two iterons. These studies show also that pi bound to a consensus site promotes occupancy of an adjacent mutated site, another hallmark of cooperative interactions. pi monomer/iteron interactions were quantified using a monomer-biased pi variant in vitro with the same collection of two-iteron constructs. The cooperativity coefficients mirror the plasmid incompatibility results for each construct tested. pi dimer/iteron interactions were quantified with a dimer-biased mutant in vitro and it was found that pi dimers bind with negligible cooperativity to two tandem iterons.     

Reversible sequestration of active site cysteines in a 2Fe-2S-bridged dimer provides a mechanism for glutaredoxin 2 regulation in human mitochondria

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-to-serine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.     

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe3(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.     

The Propeptides of VEGF-D Determine Heparin Binding,Receptor Heterodimerization,and Effects on Tumor Biology

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.     

Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins

Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency.     

Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere

Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.     

Inactivation of human fibroblast growth factor-1 (FGF-1) activity by interaction with copper ions involves FGF-1 dimer formation induced by copper-catalyzed oxidation.

Although the angiogenic proteins acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) both interact with the transition metal copper, itself a putative modulator of angiogenesis, a role for copper in FGF function has not been established. Using nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we detect the complete conversion of recombinant forms of human FGF-1 monomer protein to FGF-1 homodimers after exposure to copper ions. In contrast, not all forms of bovine FGF-1 isolated from bovine brain or a recombinant preparation of human FGF-2 completely formed homodimers after exposure to copper ions under similar conditions. Since the copper-induced FGF-1 homodimers reverted to the monomer form in the presence of dithiothreitol, specific alkylation of cysteine residues by pyridylethylation prevented FGF-1 homodimer formation, and preformed FGF-1 homodimers could not be dissociated by the metal chelator EDTA, FGF-1 dimer formation appeared to result from the formation of intermolecular disulfide bonds by copper-induced oxidation of sulfhydryl residues. FGF-1 homodimers bound with similar apparent affinity as FGF-1 monomers to immobilized copper ions, both eluting at 60 mM imidazole. Both human FGF-1 monomer and dimer forms had a 6-fold higher apparent affinity for immobilized copper ions, as compared with human FGF-2, which eluted in the monomer form at 10 mM imidazole. Further, in contrast to FGF-1 monomers, which dissociate from immobilized heparin in 1.0 M NaCl, preformed FGF-1 homodimers had reduced apparent affinity for immobilized heparin and eluted at 0.4 M NaCl. In contrast, the apparent affinity of human FGF-2 for immobilized heparin was unaffected after exposure to copper ions. Heparin appeared to modulate the formation of copper-induced intermolecular disulfide bonds for FGF-1 but not FGF-2, since co-incubation of heparin and copper with FGF-1 monomers resulted in dimers and other oligomeric complexes. FGF-1 copper-induced homodimers failed to induce mitogenesis in [3H]thymidine incorporation assays, an effect which could be reversed by treatment with dithiothreitol, whereas FGF-2-induced mitogenic activity was relatively unaffected by pretreatment with copper. The differences between human FGF-1 and FGF-2 in protein-copper interactions may be due to differing free thiol content and arrangement between the two proteins. A recombinant human FGF-1 mutant containing the two cysteines conserved throughout the FGF family of proteins but lacking a cysteine residue (Cys 131) present in wild-type human FGF-1 but not human FGF-2 readily formed copper-induced dimers.(ABSTRACT TRUNCATED AT 400 WORDS)     

The extracellular calcium-sensing receptor dimerizes through multiple types of intermolecular interactions

Recent studies have shown that the G protein-coupled, extracellular calcium ([Ca(2+)](o))-sensing receptor (CaR) forms disulfide-linked dimers through cysteine residues within its extracellular domain and that dimerization of the CaR has functional implications. In this study, we have investigated which of these disulfide linkages are essential for dimerization of the CaR and whether they are required for these functional interactions. Our results confirm the key roles of Cys(129) and Cys(131) in CaR dimerization. However, utilizing cross-linking of the CaR or immunoprecipitation of a non-FLAG-tagged CaR with a FLAG-tagged CaR using anti-FLAG antibody, we demonstrate that CaRs with or without these two cysteines form dimers on the cell surface to a similar extent. In addition, reconstitution of CaR-mediated signaling by cotransfection of two individually inactive mutant CaRs is nearly identical in the presence or absence of both Cys(129) and Cys(131), showing that covalent linkage of CaR dimers is not needed for functional interactions between CaR monomers. These findings suggest that the CaR has at least two distinct types of motifs mediating dimerization and functional interactions, i.e. covalent interactions involving intermolecular disulfide bonds and noncovalent, possibly hydrophobic, interactions.     

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.     

The small protein CP12: a protein linker for supramolecular complex assembly

CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants. It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12. The role of CP12 in this complex assembly has not been determined. To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli. Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12. On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change. This GAPDH/CP12 complex binds PRK to form a half-complex (one unit). This unit probably dimerizes due partially to interactions between the enzymes of each unit. Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions. Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured. Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly. CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants.     

Human cystatin C, an amyloidogenic protein, dimerizes through three-dimensional domain swapping

The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.     

Molecular Cloning of a Novel Vascular Endothelial Growth Factor, VEGF-D

We have identified and characterized a novel vascular endothelial growth factor (VEGF), VEGF-D, which is structurally related to vascular endothelial growth factor C. A full-length cDNA for human VEGF-D was cloned following the identification of an EST obtained through a TFASTA search of public EST databases. The murine VEGF-D was subsequently isolated from a mouse lung cDNA library. The human VEGF-D gene was mapped to human chromosome Xp22.31. Both human and mouse VEGF-D are strongly expressed in lung and encode the eight cysteine residues that are highly conserved among the members of this family. The high level of conservation between mouse and human VEGF-D may emphasize the biological importance of this gene. Recently the murine gene, FIGF, which is identical to mouse VEGF-D, was reported.     

Alteration of dark respiration and reduction of phototrophic growth in a mitochondrial DNA deletion mutant of Chlamydomonas lacking cob, nd4, and the 3' end of nd5.

We describe here a new type of mitochondrial mutation (dum24; for dark uniparental minus inheritance) of the unicellular photosynthetic alga Chlamydomonas reinhardtii. The mutant fails to grow under heterotrophic conditions and displays reduced growth under both photoautotrophic and mixotrophic conditions. In reciprocal crosses between mutant and wild-type cells, the meiotic progeny only inherit the phenotype of the mating-type minus parent, indicating that the dum24 mutation exclusively affects the mitochondrial genome. Digestion with various restriction enzymes followed by DNA gel blot hybridizations with specific probes demonstrated that dum24 cells contain four types of altered mitochondrial genomes: deleted monomers lacking cob, nd4, and the 3' end of the nd5 gene; deleted monomers deprived of cob, nd4, nd5, and the 5' end of the cox1 coding sequence; and two types of dimers produced by end-to-end fusions between monomers similarly or differently deleted. Due to these mitochondrial DNA alterations, complex I activity, the cytochrome pathway of respiration, and presumably, the three phosphorylation sites associated with these enzyme activities are lacking in the mutant. The low respiratory rate of the dum24 cells results from the activities of rotenone-resistant NADH dehydrogenase, complex II, and alternative oxidase, with none of these enzymes being coupled to ATP production. To our knowledge, this type of mitochondrial mutation has never been described for photosynthetic organisms or more generally for obligate aerobes.     

A critical role of vascular endothelial growth factor D in zebrafish embryonic vasculogenesis and angiogenesis

The VEGF family comprises seven members that are designated VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PlGF), and VEGF-F. Of these factors, VEGF-D plays important roles for angiogenesis and lymphangiogenesis, and could promote tumor growth and lymphatic metastasis. In this study, we identified a zebrafish VEGF-D homolog that encodes a 272 amino acid protein including a PDGF (platelet-derived growth factor) domain characteristic to VEGF family. Expression profile demonstrated that the VEGF-D began expressed from 13 somite stage. Microinjecting zVEGF-D mRNA into zebrafish 1-cell stage embryos resulted in severe misguidance of intersegmental vessels (ISV) and abnormal connection between dorsal aorta and caudal vein. Microangiography indicated that these abnormal ISVs were not functional. Our studies therefore identified the first non-mammalian VEGF-D and established its in vivo role for vascular system development during vertebrate embryogenesis and provided an alternative animal model to further reveal functions of VEGF-D.