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Biomedical subjects

J F Riordan

Publications and source records attributed to J F Riordan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Proteolytic release of human angiotensin-converting enzyme expressed in Chinese hamster ovary cells is enhanced by phorbol ester.

Membrane-bound angiotensin-converting enzyme (ACE) expressed in Chinese hamster ovary (CHO) cells is proteolytically released in soluble form into the medium. We find that this release is stimulated up to 50-fold by phorbol-12,13-dibutyrate and also by the addition of fresh, serum-containing media. Concomitant with the enhanced release is a marked decrease in levels of membrane-bound ACE, down to 7% of resting levels in the case of phorbol ester stimulation. Staurosporine, a protein kinase C (PKC) inhibitor, abolishes the phorbol ester effect. Kinetic analysis of the stimulated release rate indicates that it is first order, likely due to substrate depletion; calculated half times, t1/2, are 174 +/- 12 min and 40 +/- 6 min for the media-change and phorbol ester stimulated rates, respectively. Thus, release of membrane-bound ACE in CHO cells is regulated, in part, by a PKC-dependent mechanism.

Alkaloids↗
PubMed1995

Angiotensin II: biosynthesis, molecular recognition, and signal transduction.

1. Angiotensin II is a well-known vasopressive octapeptide that is the principal end-product of the renin-angiotensin system. In addition to its tonic effect on vascular smooth muscle cells, it also stimulates aldosterone secretion from the adrenals and promotes sodium reabsorption through renal tubular cells. 2. These physiological functions have been appreciated for some time, but as details of the molecular and cell biology of the angiotensin response mechanism become understood, it is increasingly apparent that the hormone has a much broader repertoire. Its functional variability is made possible by (i) different enzymatic routes for its generation, (ii) different receptors distributed in different tissues, (iii) different mechanisms for receptor regulation, and (iv) different signal transduction pathways. 3. This insight is the direct consequence of advances in pharmacology that led first to inhibitors of angiotensin converting enzyme and later to angiotensin II receptor antagonists. This review looks at the current status of angiotensin biochemistry and physiology and provides a basis for anticipation of future developments.

Angiotensin II↗
PubMed1995

LRRning the RIte of springs.

The determination of the crystal structure of the ribonuclease inhibitor-ribonuclease A complex provides exciting new insight on how the leucine-rich repeat allows a single molecule to get around the problem of inhibiting an entire family of enzymes.

Animals↗
PubMed1994

Angiogenin promotes invasiveness of cultured endothelial cells by stimulation of cell-associated proteolytic activities.

Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, is 5 times more invasive when cultured in the presence of 1 microgram of bovine angiogenin per ml than in its absence. A polyclonal anti-angiogenin antibody and alpha 2-antiplasmin neutralize the effect of angiogenin, but an angiogenin-binding protein (actin) does not. Further, this concentration of angiogenin induces a 14-fold increase in the cell-associated proteolytic activity of cultured endothelial cells, determined with a tissue-type plasminogen activator-specific peptide as the substrate. In addition, cells cultured on a three-dimensional fibrin gel in the presence of angiogenin are 3 times more capable of dissolving the gel and forming focal defects in the underlying matrix. The results indicate that angiogenin can enhance the ability of endothelial cells to digest extracellular matrix components and degrade basement membrane, thereby facilitating cell invasion and migration. Binding of angiogenin to its cell-surface binding protein (actin) followed by dissociation of the angiogenin-actin complex from the cell surface and subsequent activation of tissue-type plasminogen activator/plasmin are likely steps involved in the processes of endothelial cell invasion and angiogenesis.

Angiogenesis Inducing Agents↗
PubMed1994

Identification of the nucleolar targeting signal of human angiogenin.

Angiogenin is endocytosed by subconfluent endothelial cells, translocated to the nucleus and accumulates in the nucleolus. It also localizes into the nucleolus of digitonin-permeabilized endothelial cells. The peptide RRRGL corresponding to residues 31-35 of human angiogenin specifically targets non-nuclear carrier proteins such as albumin, an anti-human nucleolus monoclonal antibody and R33A angiogenin to the nucleolus of permeabilized endothelial cells. Proteins conjugated with a "mutant" peptide, RRAGL, are not imported. Fluorescein isothiocyanate-conjugated RRRGL is also rapidly imported into the nucleus and localized to the nucleolus, whereas the "mutant" peptide is not. Residue R33 is essential for nuclear translocation and R31 and R32 appear to modulate this process. Thus, 31RRRGL35 is a nuclear localization signal responsible for the nucleolar targeting of human angiogenin.

Amino Acid Sequence↗
PubMed1994

Crystal structure of human angiogenin reveals the structural basis for its functional divergence from ribonuclease.

Angiogenin, a potent inducer of neovascularization, is the only angiogenic molecule known to exhibit ribonucleolytic activity. Its overall structure, as determined at 2.4 A, is similar to that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor binding site, both of which are critically involved in biological function. Most strikingly, the site that is spatially analogous to that for pyrimidine binding in ribonuclease A differs significantly in conformation and is "obstructed" by glutamine-117. Movement of this and adjacent residues may be required for substrate binding to angiogenin and, hence, constitute a key part of its mechanism of action.

Amino Acid Sequence↗
PubMed1994

Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.

The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucleotide, and cyclic nucleotide substrates, but without changing specificity. The enhanced activity of Q117G toward CpA is due to a 5-fold decrease in Km and a 6-fold increase in kcat. Its Ki value for 2'-CMP is 5-fold lower than that of native angiogenin, whereas its Ki value for 5'-AMP is unchanged. It has been reported previously that mutating Asp-116 to Ala increases activity 15-fold. The double mutant D116A/Q117A is shown to be only slightly more active than each individual mutant. The present results demonstrate that Gln-117 impedes the ribonucleolytic activity of angiogenin, as predicted by x-ray crystallography. Moreover, they suggest that prior to or during catalysis angiogenin must undergo a conformational change to reorient the C-terminal segment that contains this residue, and that a similar reorganization is required for the mutants as well. This view is supported by molecular modeling of an angiogenin-uridine vanadate complex. These in vitro findings have implications for the angiogenic activity of angiogenin in vivo.

Base Sequence↗
PubMed1994

Nuclear translocation of angiogenin in proliferating endothelial cells is essential to its angiogenic activity.

The intracellular pathway of human angiogenin in calf pulmonary artery endothelial (CPAE) cells has been studied by immunofluorescence microscopy. Proliferating CPAE cells specifically endocytose native angiogenin and translocate it to the nucleus, where it accumulates in the nucleoli. Nuclear translocation of angiogenin does not occur in nonproliferative, confluent CPAE cells. These cells were previously found to express an angiogenin-binding protein (AngBP) that was identified as smooth muscle alpha-actin. Exogenous actin, an anti-actin antibody, heparin, and heparinase treatment all inhibit the internalization of angiogenin, suggesting the involvement of cell surface AngBP/actin and heparan sulfate proteoglycans in this process. It has been established that two regions of angiogenin are essential for its angiogenic activity, one is its endothelial cell binding site and the other its catalytic site capable of cleaving RNA. CPAE cells do not internalize four enzymatically active angiogenin derivatives whose cell binding site is modified, but they do internalize two enzymatically inactive mutants whose cell binding site is intact. Thus, the putative cell binding site of angiogenin is necessary for both endocytosis and nuclear translocation, but the catalytic site is not. Three other angiogenic molecules are also translocated to the nucleus of growing CPAE cells. Overall, the results suggest that nuclear translocation of angiogenin and other angiogenic molecules is a critical step in the process of angiogenesis.

Actins↗
PubMed1994

Angiotensin converting enzyme inhibitors and alcohol abuse.

Angiotensin converting enzyme plays a key role in the regulation of blood pressure and inhibitors of the enzyme are effective antihypertensive agents. An association between hypertension and alcohol abuse has long been recognized and manipulations of the renin-angiotensin system in laboratory animals has been shown to alter their consumption of ethanol. Procedures that decrease the renin-angiotensin system increase ethanol consumption. Paradoxically, inhibitors of angiotensin converting enzyme also diminish drinking. Several possible explanations for this observation have been proposed. However, observations on the relationship between stress-induced drinking and the antidipsogenic action of a fragment of adrenocorticotropic hormone suggest another possibility: angiotensin converting enzyme may be involved in the metabolism of this peptide and thereby exert an influence on drinking behavior.

Alcohol Drinking↗
PubMed1993

Angiogenin enhances actin acceleration of plasminogen activation.

Angiogenin interacts with actin to form a complex, which like actin itself, can accelerate plasmin generation by tissue plasminogen activator (tPA). In contrast to actin, the angiogenin-actin complex does not inhibit plasmin activity. In the presence of the angiogenin-actin complex, the overall proteolytic activity of a mixture of plasminogen and tPA is 11-fold higher than in its absence and 6-fold higher than in the presence of actin alone. These results suggest that binding and displacement of cell surface actin by angiogenin might be involved in cell migration and tumor invasion, processes that depend on the proteolytic degradation of basement membrane and the extracellular matrix. Therefore, the activity of the angiogenin-actin complex reported here may have physiological and pathological significance in the process of angiogenin-induced angiogenesis.

Actins↗
PubMed1993

Modulation of the activity of angiogenin by mutagenesis at Asp-116.

Substitution of Asn, Ala or His for Asp-116 in angiogenin increases its ribonucleolytic activity towards tRNA and, at least in the case of His, its ability to induce blood-vessel formation (Harper, J.W. and Vallee, B.L. (1988) Proc. Natl. Acad. Sci. USA 85, 7139-7143). Six additional Asp-116 mutants have been examined to further probe the basis for this phenomenon. Substitution of Val, Lys, Glu, or Ser increases activity towards tRNA 2-, 4-, 9- and 16-fold, respectively, whereas substitution of Trp and Pro leads to 2- and 10-fold decreases, respectively. Similar changes are seen in activity towards rRNA. Studies of base-cleavage specificity towards dinucleotide substrates (NpN') reveal a change in preference for G vs. A at the N' position when Ser replaces Asp-116 and a diminished preference for C vs. U at the N position. The Pro, Lys and Glu mutants have essentially unchanged angiogenic activity. The results demonstrate that the principal effect of replacing Asp-116 in angiogenin is to modulate enzymatic activity, possibly through an effect on His-114, and suggest that Asp-116 plays a role in controlling specificity.

Amino Acid Sequence↗
PubMed1993

Actin is a surface component of calf pulmonary artery endothelial cells in culture.

An angiogenin binding protein isolated previously from endothelial cells has been shown to be a member of the actin family. Calf pulmonary artery endothelial (CPAE) cells were investigated for the presence of surface actin by immunoblotting of isolated surface proteins and by immunofluorescence. CPAE cell surface proteins were isolated by selective apical biotinylation and recovery of biotinylated proteins by avidin affinity chromatography. Immunoblotting with a specific smooth muscle alpha-actin antibody detected the presence of this type of actin among the isolated cell surface proteins. Immunofluorescence confirmed that smooth muscle alpha-actin is localized at the surface of nonpermeabilized CPAE cells. Exposure of CPAE cells to angiogenin prior to cell surface immunostaining diminished the signal. When CPAE and rat aortic smooth muscle cells were made permeable before staining, stress fibers could be recognized by the antibody in smooth muscle cells but not CPAE cells. The results indicate that a smooth muscle type of alpha-actin is localized specifically on the surface of cultured CPAE cells where it might interact with angiogenin and other actin binding proteins present in the extracellular environment.

Actins↗
PubMed1993

Alteration of the enzymatic specificity of human angiogenin by site-directed mutagenesis.

The molecular basis for the enzymatic specificity of human angiogenin has been investigated by site-directed mutagenesis of Thr-44, Glu-108, and Ser-118--residues corresponding to those thought to be involved in substrate base recognition in the homologous protein, RNase A. Mutations of Thr-44 to Ala, His, and Asp affect both activity and specificity dramatically. The Ala and His replacements decrease activity toward tRNA by factors of 25 and 40, respectively, and reduce cleavage of cytidylyl more than uridylyl dinucleotides. Substitution by Asp does not influence the rate of tRNA and rRNA degradation but alters specificity even more markedly than the other mutations: T44D-angiogenin has 17-40-fold decreased activity toward CpN' dinucleotides and 1.3-1.9-fold increased activity toward UpN', resulting in an inverted order of preference (U > C) compared to native angiogenin. Mutations of Glu-108 to Lys and Gln change activity toward RNA and dinucleotides by no more than 50% and produce slight increases in preference for adenosine vs guanosine at position N' of NpN' substrates. Mutations of Ser-118 to Asp and Arg have a larger effect, decreasing activity by factors of approximately 2 and 4, respectively, toward all substrates examined. These results indicate that: (i) Thr-44 is important for recognition of the pyrimidine moiety at position N, (ii) Glu-108 may make a small contribution to binding the N'-nucleotide, and (iii) Ser-118 has a minor functional role, which appears to involve catalysis rather than nucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenesis Inducing Agents↗
PubMed1993

Actin is a binding protein for angiogenin.

The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the polymerization of actin below the critical concentration for spontaneous polymerization. The interaction occurs both in solution and on a poly(vinylidene difluoride) membrane. It is inhibited by excess unlabeled angiogenin and also by platelet factor 4 and protamine, which are known inhibitors of angiogenesis. Two other angiogenic molecules, basic fibroblast growth factor and tumor necrosis factor alpha, bind to 125I-labeled actin and can be crosslinked by a water-soluble carbodiimide. Both actin and an anti-actin antibody inhibit the angiogenic activity of angiogenin in the chicken embryo chorioallantoic membrane assay. The results indicate that the angiogenin binding protein is a cell surface actin and suggest that the reaction between angiogenin and this actin is an essential step in the angiogenesis process induced by angiogenin.

Actins↗
PubMed1993

Thoracoscopic biopsy in the diagnosis of pulmonary Kaposi's sarcoma.

Thoracoscopic lung biopsy provided diagnostic histologic material with minimal patient discomfort in an HIV-positive man with diffuse pulmonary Kaposi's sarcoma. This minimally invasive procedure will have an increasing role in providing a histologic diagnosis in patients with diffuse lung disease.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Crystallization and preliminary X-ray analysis of human angiogenin.

Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222(1) (a = 83.36 A, b = 120.64 A, c = 37.72 A) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 A resolution and are suitable for three-dimensional X-ray structural analysis.

Crystallization↗