PubMed Health⌕ Search

Biomedical subjects

R Volk

Publications and source records attributed to R Volk.

At least 19 recordsLinked to original sources
PubMed2006

Transport of magnetic field by a turbulent flow of liquid sodium.

We study the effect of a turbulent flow of liquid sodium generated in the von Kármán geometry, on the localized field of a magnet placed close to the frontier of the flow. We observe that the field can be transported by the flow on distances larger than its integral length scale. In the most turbulent configurations, the mean value of the field advected at large distance vanishes. However, the rms value of the fluctuations increases linearly with the magnetic Reynolds number. The advected field is strongly intermittent.

Journal Article↗
PubMed2006

Induction, helicity, and alpha effect in a toroidal screw flow of liquid gallium.

We investigate experimentally induction mechanisms in a screw flow of gallium in a toroidal channel. The flow is nonstationary and operated in a spin-down regime: the channel (and fluid) are initially set into solid body rotation; as the channel is stopped the fluid is set into strong helical motion by diverters located inside the channel. In this study, we put a particular emphasis on the induction generated by these helical motions, which are expected to develop over the entire range of turbulent scales. We apply an external magnetic field either perpendicular to the channel axis parallel to it. At large scales the nonlinear induction mechanisms are associated with the Parker stretch and twist effect and with the expulsion due to overall rotation. Induction mechanisms can also originate in the small scale helicity as in the alpha induction effect of mean-field magnetohydrodynamics. Our measurements yield an upper bound for the alpha coefficient, significantly lower than estimates based on dimensional analysis. We discuss the consequences of our observations for the engineering of homogeneous dynamos in the laboratory.

Journal Article↗
PubMed2000

Anti-angiogenic cues from vascular basement membrane collagen.

Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important antiangiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the alpha1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the alpha1beta1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.

Animals↗
PubMed2000

PR39, a peptide regulator of angiogenesis.

Although tissue injury and inflammation are considered essential for the induction of angiogenesis, the molecular controls of this cascade are mostly unknown. Here we show that a macrophage-derived peptide, PR39, inhibited the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1alpha protein, resulting in accelerated formation of vascular structures in vitro and increased myocardial vasculature in mice. For the latter, coronary flow studies demonstrated that PR39-induced angiogenesis resulted in the production of functional blood vessels. These findings show that PR39 and related compounds can be used as potent inductors of angiogenesis, and that selective inhibition of hypoxia-inducible factor-1alpha degradation may underlie the mechanism of inflammation-induced angiogenesis.

Animals↗
PubMed2000

Inhibition of ubiquitin-proteasome pathway-mediated I kappa B alpha degradation by a naturally occurring antibacterial peptide.

Induction of NF-kappaB-dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the alpha 7 subunit of the 26S proteasome and blocks degradation of NF-kappa B inhibitor I kappa B alpha by the ubiquitin-proteasome pathway without affecting overall proteasome activity. I kappa B alpha phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of I kappa B alpha degradation abolishes induction of NF-kappa B-dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-kappa B-dependent gene expression for therapeutic purposes.

Animals↗
PubMed1999

The role of syndecan cytoplasmic domain in basic fibroblast growth factor-dependent signal transduction.

To determine the role played by syndecan-4 cytoplasmic domain in the mediation of basic fibroblast growth factor (bFGF) signaling, immortalized human cells (ECV) were used to generate cell lines expressing constructs encoding full-length sequences for syndecan-4 (S4), syndecan-1 (S1), glypican-1 (G1), or chimeric proteins consisting of the ectoplasmic domain of glypican-1 linked to the transmembrane/cytoplasmic domain of syndecan-4 (G1-S4c) and the ectoplasmic domain of syndecan-4 linked to the glypican-1 glycosylphosphatidylinositol (GPI) anchor sequence (S4-GPI). Vector-transduced cells (VC) were used as controls. Expression of all these proteoglycans (except for the vector control) significantly increased cell-associated heparan sulfate mass and the number of low affinity bFGF-binding sites. However, in low serum medium, the addition of bFGF stimulated growth and migration of cells expressing S4 and G1-S4c constructs but not G1, S1, S4-GPI, or VC cells. Similar results were obtained using Matrigel growth assays. Mutations of heparan sulfate attachment sites on S4 construct abolished syndecan-4-dependent augmentation of bFGF responses. We conclude that cytoplasmic tail of syndecan-4 plays an important role in bFGF-mediated signal transduction.

Binding Sites↗
PubMed1999

Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.

Angiostatin which contains the first four kringle domains of plasminogen has been documented to be a potent inhibitor of angiogenesis. More recently, another kringle structure within plasminogen but outside angiostatin, known as kringle 5 (K5), was found to inhibit endothelial cell proliferation and migration. Here, we report the cloning and expression of mouse kringle 5 (rK5) in a bacterial expression system. The protein was purified to homogeneity using a Ni-NTA column. rK5 inhibited both proliferation and migration of endothelial cells with ED50's of 10 nM and < 500 nM, respectively. In addition, we show for the first time that rK5 causes cell cycle arrest and apoptosis, shedding further insight into rK5's mechanism of action. Finally, we show that these actions are endothelial cell specific.

Amino Acid Sequence↗
PubMed1999

Cloning, expression, and in vitro activity of human endostatin.

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we have expressed human endostatin in a yeast expression system (10 mg/L). The recombinant protein was expressed in a soluble form and purified to homogeneity. It specifically inhibited the proliferation and migration of endothelial cells. In addition, we report for the first time that endostatin caused G1 arrest of endothelial cells. Also, we show that endostatin treatment resulted in apoptosis of HUVE and HMVE cells and that all of these effects do not occur in nonendothelial cells. Collectively, these findings demonstrate the expression of a biologically active form of human endostatin in yeast and provide important mechanistic insight into endostatin action on endothelial cells.

Animals↗
PubMed1999

Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin.

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model.

Amino Acid Sequence↗
PubMed1999

Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma.

Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII that inhibits the growth of several primary tumors. We report here the cloning and expression of mouse endostatin in both prokaryotic and eukaryotic expression systems. Soluble recombinant protein expressed in yeast (15-20 mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic fibroblast growth factor. A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems. Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays. Systemic administration of endostatin at 10 mg/kg suppressed the growth of renal cell cancer in a nude mouse model. The inhibition of tumor growth with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria. In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity. Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent. Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor growth suppression by angiogenesis inhibitors.

Animals↗
PubMed1997

Macrophage-dependent regulation of syndecan gene expression.

Heparan sulfates in the extracellular matrix are required for a variety of biological processes, including cellular response to heparin-binding growth factors. However, little is known regarding the regulation of their expression and composition under pathophysiological conditions. In the present study, we have investigated the regulation of expression of two key heparan sulfate chain-carrying core proteins, syndecan-1 and syndecan-4, in a mouse/rat infarct model of tissue injury and repair. Induction of myocardial infarction was associated with a prompt increase in expression of both syndecan genes. Although infiltrating macrophages accounted for a substantial increase in syndecan expression, increased expression was noted in the levels of syndecan-1 mRNA in endothelial cells and syndecan-4 mRNA in cardiac myocytes. This increase in expression was limited to the immediate peri-infarct region and was absent from remote areas of the left or right ventricles. The influx of blood-derived macrophages in the heart correlated with the appearance of PR-39 peptide, which has previously been shown to increase syndecan expression in vitro. Studies in the op/op mice strain (which demonstrates sharply reduced levels of circulating monocytes) showed that myocardial infarction was associated with markedly reduced levels of macrophage influx and corresponding reduction in the expression of PR-39 and both syndecan genes. Pretreatment of op/op mice with granulocyte macrophage colony-stimulating factor restored myocardial macrophage content with corresponding restoration of PR-39/syndecan expression. In summary, myocardial infarction is associated with a distinct spatial and temporal pattern of syndecan-1 and -4 gene expression, which is induced by an influx of blood-derived macrophages.

Animals↗
PubMed1995

Biosynthesis of riboflavin. Studies on the reaction mechanism of 6,7-dimethyl-8-ribityllumazine synthase.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits surrounding a core of 3 alpha subunits. The beta subunits catalyze the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with (3S)-3,4-dihydroxy-2-butanone under formation of 6,7-dimethyl-8-ribityllumazine. This intermediate is converted to riboflavin by the alpha subunits via an unusual dismutation yielding 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione as second product. (3R)- and (3S)-3,4-dihydroxy-2-butanone 4-phosphate were synthesized. Both enantiomers can serve as substrate for 6,7-dimethyl-8-ribityllumazine synthase. The reaction rate of the natural S-enantiomer is about 6-fold higher than that of the R-enantiomer. The Km value for (3S)-3,4-dihydroxy-2-butanone 4-phosphate is 130 microM, and the Km value for the pyrimidine substrate is 5 microM. Diacetyl and 3,4-dihydroxy-2-butanone 3-phosphate do not serve as substrates for lumazine synthase. The enzyme-catalyzed condensation of the carbohydrate with the pyrimidine is strictly regiospecific. The enzyme does not catalyze the exchange of protons between (3S)-3,4-dihydroxy-2-butanone 4-phosphate and solvent water in the absence of the pyrimidine cosubstrate. A reaction mechanism starting with the formation of a Schiff base followed by elimination of phosphate and cyclization is proposed. The lumazine synthase activities of the native enzyme complex and of reconstituted, hollow beta 60 capsids are virtually identical (about 12,000 nmol mg-1 h-1).

Bacillus subtilis↗
PubMed1994

Genomic structure and chromosomal location of the rat gene encoding the zinc finger transcription factor Kid-1.

We have previously cloned and sequenced a novel zinc finger cDNA, Kid-1, from the rat. Because of its developmentally regulated expression pattern and its suppression after renal injury, as well as its kidney-predominant expression, we propose that Kid-1 is likely to play an important role in renal gene regulation. Kid-1 encodes a predicted protein with 13 zinc fingers at the carboxy end and Krüppel-associated box (KRAB) A and B regions at the amino terminus. Expression of a Kid-1-GAL4 chimeric protein results in strong transcriptional repression of cotransfected constructs containing GAL4 binding sites and a chloramphenicol acetyl transferase gene driven by either a minimal promoter or a SV40 enhancer. We now report the cloning, structural organization, and chromosomal localization of the Kid-1 gene. The Kid-1 gene is composed of four exons and three introns, closely reflecting the organization of the Kid-1 protein. The KRAB A and B regions are encoded by the second and third exons, respectively. The entire zinc finger region is encoded by the fourth exon. Using a combination of linkage analysis and somatic cell hybrid analysis, Kid-1 was mapped to rat chromosome (RNO) 10. Kid-1, Il3, and Sparc form a tight linkage group on RNO10. Regional sublocalization to RNO10q21.3-q22 was established by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

Differential induction of eicosanoid synthesis in monocytic cells treated with retinoic acid and 1,25-dihydroxy-vitamin D3.

Synthesis and release of eicosanoids is characteristic to mature monocytes, while in undifferentiated premonocytic cells arachidonic acid (AA) metabolism is barely detectable. Since some vitamins and cytokines induce differentiation of these cells to a more monocyte-like cell type, we evaluated the effect of retinoic acid (RA), 1,25(OH)2-vitamin D3 (1,25-D3) and interferon-gamma (IFN-gamma) on AA metabolism in the human premonocytic cell lines U937 and THP-1. In U937 cells, differentiation with RA (1 microM) followed by stimulation with the calcium ionophore A23187 (10 microM) or platelet activating factor (PAF; 100 nM) significantly increased synthesis of immunoreactive 6-keto PGF1 alpha, TxB2 and PGE2 5- to 6-fold. Analysis of AA metabolism in RA-differentiated cells prelabelled with [3H]-AA revealed the formation of additional radioactive compounds which coeluted with standards for PGF2 alpha, PGD2 and 12-hydroxyheptadecatrienoic acid (HHT). The structural identity of 6-keto PGF1 alpha, TxB2, PGE2 and HHT was confirmed by gas chromatography-mass spectrometry (GC-MS). In parallel, RA induced the expression of the monocytic surface antigen CD11b, but not CD14. Differentiation with 1,25-D3 (10 nM) only marginally increased stimulated eicosanoid formation, while it more effectively induced expression of CD11b and CD14. Pretreatment with IFN-gamma (100 IU/ml) slightly increased stimulus-dependent AA metabolism, but did not induce expression of CD11b or CD14. Induction of eicosanoid synthesis by RA was further confirmed in THP-1 cells. These data indicate that RA most effectively induced cyclooxygenase activity and stimulus-dependent eicosanoid formation in U937 and THP-1 cells. Furthermore, since expression of monocytic surface antigens differed between RA and 1,25-D3, it is suggested that induction of cyclooxygenase activity may correlate to differentiation into distinct monocytic phenotypes.

Antigens, Surface↗
PubMed1993

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.

Amino Acid Sequence↗
PubMed1992

Biosynthesis of riboflavin: cloning, sequencing, and expression of the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli.

3,4-Dihydroxy-2-butanone 4-phosphate is biosynthesized from ribulose 5-phosphate and serves as the biosynthetic precursor for the xylene ring of riboflavin. The gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase of Escherichia coli has been cloned and sequenced. The gene codes for a protein of 217 amino acid residues with a calculated molecular mass of 23,349.6 Da. The enzyme was purified to near homogeneity from a recombinant E. coli strain and had a specific activity of 1,700 nmol mg-1 h-1. The N-terminal amino acid sequence and the amino acid composition of the protein were in agreement with the deduced sequence. The molecular mass as determined by ion spray mass spectrometry was 23,351 +/- 2 Da, which is in agreement with the predicted mass. The previously reported loci htrP, "luxH-like," and ribB at 66 min of the E. coli chromosome are all identical to the gene coding for 3,4-dihydroxy-2-butanone 4-phosphate synthase, but their role had not been hitherto determined. Sequence homology indicates that gene luxH of Vibrio harveyi and the central open reading frame of the Bacillus subtilis riboflavin operon code for 3,4-dihydroxy-2-butanone 4-phosphate synthase.

Amino Acid Sequence↗
PubMed1991

Biosynthesis of riboflavin. Studies on the mechanism of L-3,4-dihydroxy-2-butanone 4-phosphate synthase.

The riboflavin precursor, L-3,4-dihydroxy-2-butanone 4-phosphate, is formed from D-ribulose 5-phosphate by a single 24-kDa enzyme. Studies with various specifically 13C-labeled D-ribulose 5-phosphates as substrate showed that the carbon atoms 1-3 of the enzyme product correspond to carbon atoms 1-3 of the substrate, whereas C-4 of the product stems from C-5 of the substrate. Carbon atom 4 of the substrate is released as formate together with the hydrogen atom attached to it. The skeletal rearrangement which leads to the loss of C-4 and the direct linkage between C-3 and C-5 of the substrate is an intramolecular reaction. The hydrogen atom at C-3 of the enzyme product is introduced from solvent water. A reaction mechanism which is in agreement with all experimental data is proposed.

Candida↗