[Vascular development and molecular regulation of angiogenesis during tumorigenesis].
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Animals↗
Biomedical subjects
H Sun
Publications and source records attributed to H Sun.
Effect of cellular filamentation on adventurous and social gliding motility of Myxococcus xanthus.
Filamentous bacterial cells often provide biological information that is not readily evident in normal-size cells. In this study, the effect of cellular filamentation on gliding motility of Myxococcus xanthus, a Gram-negative social bacterium, was investigated. Elongation of the cell body had different effects on adventurous and social motility of M. xanthus. The rate of A-motility was insensitive to cell-body elongation whereas the rate of S-motility was reduced dramatically as the cell body got longer, indicating that these two motility systems work in different ways. The study also showed that filamentous wild-type cells glide smoothly with relatively straight, long cell bodies. However, filamentous cells of certain social motility mutants showed zigzag, tangled cell bodies on a solid surface, apparently a result of a lack of coordination between different fragments within the filaments. Further genetic and biochemical analyses indicated that the uncoordinated movements of these mutant filaments were correlated with the absence of cell surface fibril materials, indicating a possible new function for fibrils.
Cell Wall↗
PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments.
We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5'-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin betagamma subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.
Alternative Splicing↗
Nonessential role for methionines in the productive association between calmodulin and the plasma membrane Ca-ATPase.
To investigate the role of hydrophobic interactions involving methionine side chains in facilitating the productive association between calmodulin (CaM) and the plasma membrane (PM) Ca-ATPase, we have substituted the polar amino acid Gln for Met at multiple positions in both the amino- and carboxyl-terminal domains of CaM. Conformationally sensitive fluorescence signals indicate that these mutations have little effect on the backbone fold of the carboxyl-terminal domain of CaM. The insertion of multiple Gln in either globular domain results in a decrease in the apparent affinity of CaM for the PM-Ca-ATPase. However, despite the multiple substitution of Gln for four methionines at positions 36, 51, 71, and 72 in the amino-terminal domain or for three methionines at positions 124, 144, and 145 in the carboxyl-terminal domain, these mutant CaMs are able to fully activate the PM-Ca-ATPase. Thus, although these CaM mutants have a decreased affinity for the CaM-binding site on the Ca-ATPase, they retain the ability to fully activate the Ca-ATPase at saturating concentrations of CaM. The role of individual methionines in modulating the affinity between the carboxyl terminus and the PM-Ca-ATPase was further investigated through the substitution of individual Met with Gln. Upon substitution of Met(124) and Met(144) with Gln, there is a 5- and 10-fold increase in the amount of CaM necessary to obtain half-maximal activation of the PM-Ca-ATPase, indicating that these methionine side chains participate in the high-affinity association between CaM and the PM-Ca-ATPase. However, substitution of Gln for Met(145) results in no change in the apparent affinity between CaM and the PM-Ca-ATPase, indicating that in contrast to all other known CaM targets, Met(145) does not participate in the interaction between CaM and the PM-Ca-ATPase. These results emphasize differences in the binding interactions between individual methionines in CaM and different target enzymes, and suggest that hydrophobic interactions between methionines in CaM and the binding site on the PM-Ca-ATPase are not necessary for enzyme activation. Calculation of the binding affinities of individual CaM domains associated with activation of the PM-Ca-ATPase suggests that mutations of methionines located in either domain of CaM can decrease the initial high-affinity association between CaM and the PM-Ca-ATPase, but have little effect upon the subsequent binding of the opposing globular domain. These results suggest that the initial associations between CaM and the CaM-binding sequence in the PM-Ca-ATPase are guided by nonspecific hydrophobic interactions involving both domains of CaM.
Amino Acid Substitution↗
Interactions of bismuth complexes with metallothionein(II).
Bismuth complexes are widely used as anti-ulcer drugs and can significantly reduce the side effects of platinum anti-cancer drugs. Bismuth is known to induce the synthesis of metallothionein (MT) in the kidney, but there are few chemical studies on the interactions of bismuth complexes with metallothionein. Here we show that Bi(3+) binds strongly to metallothionein with a stoichiometry bismuth:MT = 7:1 (Bi(7)MT) and can readily displace Zn(2+) and Cd(2+). Bismuth is still bound to the protein even in strongly acidic solutions (pH 1). Reactions of bismuth citrate with MT are faster than those of [Bi(EDTA)](-), and both exhibit biphasic kinetics. (1)H NMR data show that Zn(2+) is displaced faster than Cd(2+), and that both Zn(2+) and Cd(2+) in the beta-domain (three metal cluster) of MT are displaced by Bi(3+) much faster than from the alpha-domain (four metal cluster). The extended x-ray absorption fine structure spectrum of Bi(7)MT is very similar to that for the glutathione and N-acetyl-L-cysteine complexes [Bi(GS)(3)] and [Bi(NAC)(3)] with an inner coordination sphere of three sulfur atoms and average Bi-S distances of 2.55 A. Some sites appear to contain additional short Bi-O bonds of 2.2 A and longer Bi-S bonds of 3.1 A. The Bi(3+) sites in Bi(7)MT are therefore highly distorted in comparison with those of Zn(2+) and Cd(2+).
Acetylcysteine↗
Calcium-dependent structural coupling between opposing globular domains of calmodulin involves the central helix.
We have used fluorescence spectroscopy to investigate the average structure and extent of conformational heterogeneity associated with the central helix in calmodulin (CaM), a sequence that contributes to calcium binding sites 2 and 3 and connects the amino- and carboxyl-terminal globular domains. Using site-directed mutagenesis, a double mutant was constructed involving conservative substitution of Tyr(99) --> Trp(99) and Leu(69) --> Cys(69) with no significant effect on the secondary structure of CaM. These mutation sites are at opposite ends of the central helix. Trp(99) acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements of the conformation of the central helix. Cys(69) provides a reactive group for the covalent attachment of the FRET acceptor 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS). AEDANS-modified CaM fully activates the plasma membrane (PM) Ca-ATPase, indicating that the native structure is retained following site-directed mutagenesis and chemical modification. We find that the average spatial separation between Trp(99) and AEDANS covalently bound to Cys(69) decreases by approximately 7 +/- 2 A upon calcium binding. However, irrespective of calcium binding, there is little change in the conformational heterogeneity associated with the central helix under physiologically relevant conditions (i.e., pH 7.5, 0.1 M KCl). These results indicate that calcium activation alters the spatial arrangement of the opposing globular domains between two defined conformations. In contrast, under conditions of low ionic strength or pH the structure of CaM is altered and the conformational heterogeneity of the central helix is decreased upon calcium activation. These results suggest the presence of important ionizable groups that affect the structure of the central helix, which may play an important role in mediating the ability of CaM to rapidly bind and activate target proteins.
Amino Acid Substitution↗
Iron isotope biosignatures.
The (56)Fe/(54)Fe of Fe-bearing phases precipitated in sedimentary environments varies by 2.5 per mil (delta(56)Fe values of +0.9 to -1. 6 per mil). In contrast, the (56)Fe/(54)Fe of Fe-bearing phases in igneous rocks from Earth and the moon does not vary measurably (delta(56)Fe = 0.0 +/- 0.3 per mil). Experiments with dissimilatory Fe-reducing bacteria of the genus Shewanella algae grown on a ferrihydrite substrate indicate that the delta(56)Fe of ferrous Fe in solution is isotopically lighter than the ferrihydrite substrate by 1.3 per mil. Therefore, the range in delta(56)Fe values of sedimentary rocks may reflect biogenic fractionation, and the isotopic composition of Fe may be used to trace the distribution of microorganisms in modern and ancient Earth.
Earth, Planet↗
Transferrin as a metal ion mediator.
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Journal Article↗
Simultaneous multisite mapping of the right and the left atrial septum in the canine intact beating heart.
BACKGROUND: The spread of activation between the right atrium (RA) and left atrium (LA), particularly along the right and left aspects of the interatrial septum, is not clear. METHODS AND RESULTS: Basket-shaped catheters carrying 64 electrodes were deployed into both the RA and LA of 10 dogs. Position and orientation of the baskets were determined by fluoroscopy and echocardiography. Basket unipolar electrograms were simultaneously recorded in each dog during sinus rhythm, right ventricular pacing, and pacing of the right septum through the basket in the superior and inferior regions. Isochrone maps depicting all aspects of the atria, including the septum, were compared. During sinus rhythm and superior right septal pacing, wave fronts propagated predominantly from superior to inferior regions on both the right and left septum. However, activation of the left septum was delayed compared with the right septum. During right ventricular pacing and inferior right septal pacing, activation of the septum was discordant; 1 wave front propagated rapidly on the right septum from inferior to superior regions, whereas 2 opposing wave fronts originated on the left septum in both the superior and inferior regions. The left septum was activated predominantly by the superior wave front. Activation of the left septum was completed in a significantly shorter time during pacing of the right septum in the inferior region compared with the superior region. CONCLUSIONS: In dogs, activation of the right and left aspects of the interatrial septum is discordant. Electrical connections are present between the RA and LA in regions superior as well as inferior to the septum.
Animals↗
Molecular modeling and synthesis of inhibitors of herpes simplex virus type 1 uracil-DNA glycosylase.
We recently reported the properties of the first selective inhibitors of herpes simplex virus type 1 (HSV1) uracil-DNA glycosylase (UDG), an enzyme of DNA repair that has been proposed to be required for reactivation of the virus from latency. 6-(4-Octylanilino)uracil (octAU) was the most potent inhibitor among a series of 6-(4-alkylanilino)uracils, acting in the micromolar range and without effect against human UDG. A 28.5-kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. We have used the coordinates of this structure in order to study interaction of our inhibitors with the enzyme, and a model of binding between octAU and UDG has been derived. Starting with the optimized model, the activity of several octAU analogues was predicted, and the values compared favorably with experimental results found for the synthetic compounds. Several hydrophilic derivatives were predicted and found to be active as UDG inhibitors. These compounds will be useful to determine if UDG, like the viral thymidine kinase, is required for reactivation of HSV1 from latency in nerve cells.
Aniline Compounds↗
The PTEN tumor suppressor homolog in Caenorhabditis elegans regulates longevity and dauer formation in an insulin receptor-like signaling pathway.
Inactivation of the tumor suppressor PTEN gene is found in a variety of human cancers and in cancer predisposition syndromes. Recently, PTEN protein has been shown to possess phosphatase activity on phosphatidylinositol 3,4,5-trisphosphate, a product of phosphatidylinositol 3-kinase. We have identified a homolog of PTEN in Caenorhabditis elegans and have found that it corresponds to the daf-18 gene, which had been defined by a single, phenotypically weak allele, daf-18(e1375). By analyzing an allele, daf-18(nr2037), which bears a deletion of the catalytic portion of CePTEN/DAF-18, we have shown that mutation in daf-18 can completely suppress the dauer-constitutive phenotype caused by inactivation of daf-2 or age-1, which encode an insulin receptor-like molecule and the catalytic subunit of phosphatidylinositol 3-kinase, respectively. In addition, daf-18(nr2037) dramatically shortens lifespan, both in a wild-type background and in a daf-2 mutant background that normally prolongs lifespan. The lifespan in a daf-18(nr2037) mutant can be restored to essentially that of wild type when combined with a daf-2 mutation. Our studies provide genetic evidence that, in C. elegans, the PTEN homolog DAF-18 functions as a negative regulator of the DAF-2 and AGE-1 signaling pathway, consistent with the notion that DAF-18 acts a phosphatidylinositol 3,4,5-trisphosphate phosphatase in vivo. Furthermore, our studies have uncovered a longevity-promoting activity of the PTEN homolog in C. elegans.
Amino Acid Sequence↗
p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27.
Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27. Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.
Cell Cycle↗
Molecular basis of feline beta-glucuronidase deficiency: an animal model of mucopolysaccharidosis VII.
A family of domestic cats was found that exhibited clinical and biochemical abnormalities consistent with mucopolysaccharidosis VII, an autosomal recessive lysosomal storage disorder caused by beta-glucuronidase deficiency. beta-Glucuronidase activity was undetectable in affected cat fibroblasts and restored by retroviral gene transfer of rat beta-glucuronidase cDNA. beta-Glucuronidase mRNA was normal in affected cat testis by Northern blot analysis. Normal feline beta-glucuronidase cDNA was cloned and characterized, and amplified from affected cat fibroblasts by reverse transcription coupled polymerase chain reaction. There was a G-to-A transition in the affected cat cDNA that predicted an E351K substitution, destroyed a BssSI site, and eliminated GUSB enzymatic activity in expression studies. Multiple species comparison and the crystal structure of human beta-glucuronidase indicated that E351 is a highly conserved residue most likely essential in maintenance of the enzyme's conformation. BssSI digestion of polymerase chain reaction products amplified from genomic DNA indicated that affected cats were homozygous and cats with half-normal beta-glucuronidase activity were heterozygous for the missense mutation. Carriers identified in this manner produced affected kittens in prospective breedings, and a feline MPS VII breeding colony has been established.
Amino Acid Sequence↗
[Alterations of p16 gene in transformed BEP2D cells induced by NNK].
Silver-staining PCR-SSCP method was used to detect the alterations of p16 gene in the transformed BEP2D cells induced by NNK. Compared with control cells, the abnormal shifting of the single-stranded DNA(ssDNA) in E1. beta but not p16 Exon 1-3 was identified in transformed BEP2D cells. The results suggested that the mutation and/or deletion in p16 gene might be one cause of NNK-unduced carcinogenesis in human bronchial epichellium.
Animals↗
High affinity interactions of nucleolin with G-G-paired rDNA.
Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is KD = 1 nM. Two separate domains of nucleolin can interact with G-G-paired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-G-paired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination.
Animals↗
Marked mitigation of transplant vascular sclerosis in FasLgld (CD95L) mutant recipients. The role of alloantibodies in the development of chronic rejection.
BACKGROUND: In the acute rejection of allografts, the interaction between Fas (CD95) and its ligand (FasL; CD95L) has been shown to be involved in mediating apoptotic cell death. The role, however, of these molecules in the pathogenesis of transplant vascular sclerosis is as yet undetermined. The present study was therefore designed to address this issue. MATERIAL: C3H/HEJ FasLgld (FasL-; H2k) spontaneously mutant mice were used either as donors or recipients of aortic allografts; wild-type C57B1/6 (B6; H2b) were used as corresponding recipients or donors (n=6/group), respectively. Controls included aortas transplanted across appropriate allogeneic and syngeneic strain combinations. For histopathological evaluations, the grafts were harvested at day 40 after transplantation, at which time, splenocytes and sera were also obtained for mixed leukocyte reaction and complement-mediated microcytotoxicity assays, respectively. RESULTS: Similar to aortas obtained from allogeneic controls, allografts harvested from FasL- -->B6 recipients had morphological evidence of chronic rejection characterized by circumferential intimal thickening with partial disruption of the elastic membranes. Correspondingly, heightened antidonor cellular reactivity was also witnessed in these recipients. On the contrary, B6 allografts harvested from the majority of C3H-->FasL- recipients exhibited marked preservation of aortic morphology. Although these recipients had diminished antidonor cellular proliferation, the titers of alloantibodies were markedly elevated. CONCLUSION: The presence of FasL-expressing functional cytotoxic T cells is required for the pathogenesis of transplant vascular sclerosis. The significant reduction and/or absence of chronic rejection with the concomitant retention of antidonor humoral response in C3H FasL- recipients of B6 aortas prompt us to suggest that perhaps posttransplantation vasculopathy is initiated by cell-mediated cytotoxicity with its perpetuation facilitated by alloantibodies.
Animals↗
PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.
To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten-/- ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G1/S transition was accompanied by down-regulation of p27(KIP1), a major inhibitor for G1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten-/- cell survival. Our studies suggest that PTEN regulates the phosphatidylinositol 3,4, 5,-trisphosphate and Akt signaling pathway and consequently modulates two critical cellular processes: cell cycle progression and cell survival.
Animals↗
Retrovirus vector-mediated correction and cross-correction of lysosomal alpha-mannosidase deficiency in human and feline fibroblasts.
Lysosomal alpha-mannosidase (EC 3.2.1.24) is an exoglycosidase in the glycoprotein degradation pathway. A deficiency of this enzyme causes the lysosomal storage disease alpha-mannosidosis. Retrovirus vector transfer of a new human alpha-mannosidase cDNA resulted in high-level expression of alpha-mannosidase enzymatic activity in deficient human and feline fibroblasts. The expressed alpha-mannosidase had the same biochemical properties (thermal stability, pH profile, inhibitor/activator sensitivity) as the native enzyme expressed in normal cells. The transferred enzyme colocalized with a control lysosomal hydrolase in cell fractionation experiments. The vector-encoded enzyme also was released at high levels from the corrected cells, and was taken up by untreated mutant cells via the mannose 6-phosphate receptor-mediated endocytic pathway (cross-correction). It is envisioned that genetic correction of a subset of cells (e.g., hematopoietic stem cells) in patients will provide a source of corrective enzyme for other affected tissues in this multisystem disease. Development of a vector expressing high levels of alpha-mannosidase that cross-corrects mutant cells will enable somatic gene transfer experiments in the cat model of human alpha-mannosidosis.
Animals↗