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A Mironov

Publications and source records attributed to A Mironov.

At least 37 records · Page 2Linked to original sources
PubMed1997

Role of NAD+ and ADP-ribosylation in the maintenance of the Golgi structure.

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114-1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065-7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre-ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200-14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre-ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50-enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.

Adenosine Diphosphate Ribose↗
PubMed1997

Characterization of chemical inhibitors of brefeldin A-activated mono-ADP-ribosylation.

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 microg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the Vmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+ (154 +/- 13 microM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

Adenosine Diphosphate↗
PubMed1997

Possible role of BARS-50, a substrate of brefeldin A-dependent mono-ADP-ribosylation, in intracellular transport.

Brefeldin A (BFA), a fungal metabolite that inhibits membrane transport, potently stimulates an endogenous ADP-ribosylation reaction that selectively modifies two cytosolic proteins of 38 and 50 kDa on an amino acid residue different from those used by all known mADPRTs. The 38-kDa substrate was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), whereas the 50-kDa substrate (BARS-50) was characterized as a novel guanine nucleotide binding protein. Thus, BARS-50 is able to bind GTP and its ADP-ribosylation is inhibited by the beta gamma subunit of GTP-binding (G) proteins. Moreover, BARS-50 was demonstrated to be a group of closely related proteins that appear to be different from all the known G proteins. A partially purified BARS-50 was obtained from rat brain cytosol, which was then used for microsequencing and in functional studies. A similar procedure led to the purification of native (non-ADP-ribosylated) BARS-50. The possible role of the BFA-dependent ADP-ribosylation and of BARS-50 in the maintenance of Golgi structure and function was addressed by examining which of the effects of BFA may be modified by inhibiting this reaction. We find that the BFA-dependent transformation of the Golgi stacks into a tubular reticular network is prevented when the BFA-dependent ADP-ribosylation activity was blocked by specific inhibitors thus indicating that BFA-dependent ADP-ribosylation of cytosolic proteins participate in the dynamic regulation of intracellular transport.

Adenosine Diphosphate Ribose↗
PubMed1997

Brefeldin A-induced ADP-ribosylation in the structure and function of the Golgi complex.

Brefeldin A (BFA) is a fungal metabolite that exerts generally inhibitory actions on membrane transport and induces the disappearance of the Golgi complex. Previously we have shown that BFA stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 KD. The BFA-binding components mediating the BFA-sensitive ADP-ribosylation (BAR) and the effect of BFA on ARF binding to Golgi membranes have similar specificities and affinities for BFA and its analogues, suggesting that BAR may have a role in the cellular effects of BFA. To investigate this we used the approach to impair BAR activity by the use of BAR inhibitors. A series of BAR inhibitors was developed and their effects were studied in RBL cells treated with BFA. In addition to the common ADP-ribosylation inhibitors (nicotinamide and aminobenzamide), compounds belonging to the cumarin (novobiocin, cumermycin, dicumarol) class were active BAR inhibitors. All BAR inhibitors were able to prevent the BFA-induced redistribution of a Golgi marker (Helix pomatia lectin) into the endoplasmic reticulum, as assessed in immunofluorescence experiments. At the ultrastructural level, BAR inhibitors prevented the tubular-vesicular transformation of the Golgi complex caused by BFA. The potencies of these compounds in preventing the BFA effects on the Golgi complex were similar to those at which they inhibited BAR. Altogether these data support the hypothesis that BAR mediates at least some of the effects of BFA on the Golgi structure and function.

Adenosine Diphosphate Ribose↗
PubMed1997

Characterization of the endogenous mono-ADP-ribosylation stimulated by brefeldin A.

We have recently described a novel enzymatic mono-ADP-ribosyl transfer reaction induced by brefeldin A, a well characterized inhibitor of vesicular traffic, which selectively modifies two cytosolic proteins of 38 kDa (p38) and 50 kDa (BARS-50). p38 was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme and a multifunctional protein involved in several cellular processes; BARS-50 might be a novel G protein, since it is able to bind GTP and the beta gamma subunit of G proteins. We have characterized this enzymatic activity and screened in vitro the effects of different drugs belonging to the coumarine (dicumarol, coumermicin A1 and novobiocin) and quinone (ilimaquinones, benzoquinones and naphtoquinones) class. These drugs blocked the BFA-dependent mono-ADP-ribosylation, showed remarkable effects on Golgi morphology in control cells, and antagonized the tubular reticular redistribution of the Golgi complex in brefeldin A treated cells (see papers of Corda and Colanzi in this issue) suggesting a possible role for ADP-ribosylation in both the cellular effects of brefeldin A and the maintenance of the structure/function of the Golgi complex.

Adenosine Diphosphate Ribose↗
PubMed1996

Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study.

Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled 35S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation).

Animals↗
PubMed1996

Atherosclerosis-prone branch regions in human aorta: microarchitecture and cell composition of intima.

The microarchitecture and cell composition of intima were studied at the macroscopically unaffected branch regions of human thoracic aorta using en face preparations, scanning and transmission electron microscopy, and immunohistochemistry. The endothelial lining showed a heterogeneous pattern and altered morphology including the areas of deendothelialization covered with platelets and dilated intercellular clefts. Leukocyte adhesion, accumulation of subendothelial macrophages and lymphocytes were characteristic of proximal and lateral zones, while the flow divider showed no significant accumulation of blood cells. Smooth muscle cells (SMCs) on the flow divider were elongated, in a contractile state, contacted side-by-side and did not contain lipid inclusions. In the lateral and proximal zones, intima appeared to be a network of stellate SMCs which were in contact through their processes. Most of the SMCs were in a synthetic state and many of them contained small lipid droplets. The number of procollagen I positive cells and the volume of extracellular components were most significant at the lateral zones rather than at the flow divider. We did not observe any difference in the rate of proliferation. Our results suggest that the intimal layer at the lateral and proximal zones has some distinct structural peculiarities, which provoke the development of initial atherosclerotic lesions at these sites. Such an intimal structure is probably caused by different flow patterns at these zone. However, only the totality of different morphological features exhibited in the area of altered vascular wall shear stress may be considered as a prerequisite for atherosclerotic lesions.

Adult↗
PubMed1996

Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.

Bacterial Proteins↗
PubMed1995

The morphology of valves and valve-like structures in the canine and feline thoracic duct.

The microanatomy and ultrastructure of the feline and canine thoracic duct and afferent lymphatics were studied by scanning and transmission electron microscopy. We found that the lymphatic vessels were always terminated by ostial valves of two shapes, crescent- and navicular-like, in a ratio of 4:1. Specific regulatory structures along the free edges of the valves, including marginal thickenings and buttresses, are described. The tissue and cellular organization of the valve endothelium showed distinct peculiarities, particularly in the orientation and shape of the cells and their microrelief. We found that valvular endothelial cells, especially ¿tip cells¿, which are situated in unfavourable lymphodynamic conditions, were characterized by an increased volume density of intermediate (probably vimentin-based) filaments, suggesting an accommodative mechanism involving such filaments.

Animals↗
PubMed1995

Arterial lesions in hypercholesterolaemic Yoshida rats: morphological evaluation.

The aortic wall structure of genetically determined hypercholesterolaemic (Yoshida) and control (Brown-Norway) rats was investigated by transmission and scanning electron and light microscopy. Leucocyte adherence mostly at branch sites and irregular protrusive structures were observed on the endothelial surface of the thoracic aorta of Yoshida, but not of Brown-Norway rats. The subendothelial space of the aortic wall of Yoshida rats was characterized by intimal cushions consisting of smooth muscle cells of 'synthetic phenotype' associated with adhering leucocytes and lipid droplets. Lipid infiltration of the cytoplasm of medial smooth muscle cells was observed on the inner part of the aortic arch and on the lateral parts of the large branches of Yoshida rats. This model of spontaneously hyperlipidaemic Yoshida rats is an appropriate 'moderate' injury system, which may be useful for studies of multiple risk factors for atherogenesis.

Animals↗
PubMed1995

Acquired carotid-cavernous fistula caused by traumatic intracavernous rupture of an embryonic anastomosis.

This case of traumatic carotid-cavernous fistula was caused by rupture of a peculiar ipsilateral intracavernous anastomosis between the accessory meningeal artery and the redundant deep recurrent ophthalmic artery. In the opposite cavernous sinus there was an obvious anastomosis between the accessory meningeal artery and the ophthalmic artery, probably located in the lateral part of the cavernous sinus. The patient was successfully treated with transarterial embolization followed by surgery of the cavernous sinus. In light of the vascular embryology, it is possible that unusual embryonic connections between the primitive dorsal ophthalmic artery and the accessory meningeal artery already existed in both cavernous sinus areas.

Adult↗
PubMed1995

Classification of spontaneous dural arteriovenous fistulas with regard to their pathogenesis.

PURPOSE: A classification of dural arteriovenous fistulas (DAVFs) according to their pathogenesis has not been established. The purpose of this study was to examine the hypothesis that the different angiomorphologies of DAVFs depend on the location of the venous recipient, and that a territorial classification of spontaneous DAVFs can be created based on their acquired development. MATERIAL AND METHODS: The clinical and radiographic findings of 96 patients with DAVFs were reviewed, especially with regard to their venous characteristics. RESULTS: DAVFs were subdivided into 5 groups: Type 1--DAVFs of the dural sinuses (n = 39); Type 2--DAVFs of the cavernous sinus (n = 29); Type 3--DAVFs of Galen's system (n = 10); Type 4--DAVFs of the venous plexus at the base of the skull (n = 9); Type 5--DAVFs of the parasinusal cortical veins (n = 9). The documentation of a causal sinus thrombosis depends on the location of the DAVF: in 72% of the cases with type 1 DAVFs there was a thrombosis at the time of the investigation, but no thrombosis could be proved in cases with type 4 DAVFs. CONCLUSION: The morphological development of DAVFs seems to depend on the flow volume of the venous recipient. A pronounced development of pathological AV shunts takes place at the level of the large basal dural sinuses. A delayed development of pathological AV shunts with a low shunt volume occurs in a venous recipient with a low AV pressure gradient.

Adult↗
PubMed1994

Three-dimensional organization of biological tissues: a new method for investigation using scanning electron microscopy.

A new method is described which enables the visualization of hidden cellular surfaces of biological tissues. The method does not depend on the kind of fixative solution, the duration of the fixation, or the quantity of extracellular matrix. The samples are chemically dissociated with potassium ethoxide until the first individual cells appear in the solution. This initial process is followed by phase contrast microscopy, and remaining tissue is investigated with scanning electron microscopy. The method has proved more effective than conventional enzymatic techniques and is a simple, quick, and effective way to investigate the three-dimensional organization of different normal and pathological tissues.

Aorta↗
PubMed1994

Pathogenetical consideration of spontaneous dural arteriovenous fistulas (DAVFs).

Sinus thrombosis has been regarded as an aetiological factor in DAVFs. However, this claim has been disputed in the literature; because it is not possible to prove that DAVFs arise as a result of sinus thrombosis in all cases, and there is evidence that venous thrombosis can be acquired from a DAVF. The purpose of this study is to examine the hypothesis that the different angiomorphologies of DAVFs depend on the location of their venous outflow, and that a territorial classification of spontaneous DAVFs can be created which is based on their acquired development as a consequence of the breakdown of autoregulation of dural microvascularisation after venous thrombosis. The clinical and radiographic findings of 96 patients with DAVFs were reviewed. The angiomorphology of these lesions was examined using extensive prospective and retrospective angiographic studies, especially with regard to their venous characteristics. The associated phlebothrombosis of the venous outflow was determined in all cases by angiography, and in 8 cases by CT or MRI. We also applied the Venturi effect, a well-confirmed engineering principle, to propose a new hypothesis concerning the development of acquired DAVFs: namely, that there is an acceleration of the shunt volume of the physiological dural AVshunts afer a breakdown of their autoregulation occurs. This acceleration seems to be proportional to the flow volume of the venous outflow. The imaging examination showed venous thrombosis in 51 cases. In 98% (44) of the non-thrombotic cases, predisposing histories for phlebothrombosis were documented-for example, hypercoagulable states (venous embolism, phlebothrombosis of the leg, cardiac disease), diabetes mellitus, chronic otitis media, or various low-grade infections. In addition, in 78% of the non-thrombotic cases (excluding the DAVFs of the venous plexus at the base of the skull) there were also angiographically documented deformations of the sinus wall, including hypoplasia of the sigmoid or transverse sinus (20 cases), segmental narrowing of the sinus (8 cases), septation of the sinus lumen (2 cases), and cavernous nodules projecting into the lumen of the transverse (6 cases), sigmoid (4 cases), or sagittal (3 cases) sinuses. The clinical presentation suggested that DAVFs probably develop after a breakdown of the autoregulation of the physiological AV shunts resulting from a venous thrombosis, when there are suitable arteriovenous pressure gradients in the venous recipient. This development would depend on the haemodynamic action of the Venturi effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1994

Effect of cholesterol-supplemented diet in heritable hyperlipidemic Yoshida rats: functional and morphological characterization of thoracic aorta.

In this study we have considered the possibility of inducing vascular damage in Yoshida Pittsburg (YOS) rat, an inbred strain which has endogenous hyperlipidemia without vascular atherosclerotic damage. Cholesterol-enriched diet (4% cholesterol plus 1% cholic acid and 0.5% thiouracil) was administered to YOS rats, in order to induce atherogenesis. The results indicate that, despite significant increase in serum (about 2-fold) and aortic tissue cholesterol (about 6-fold), no morphological damage occurred. A reduction in acetylcholine-mediated relaxation (of about 37%) was observed. No inhibition of ATP- or sodium nitrite-induced relaxation, or of contraction induced by norepinephrine was seen. Serum triglyceride concentration did not vary after administration of a cholesterol-enriched diet. Our results suggest that in heritable hyperlipidemic Yoshida rat, after 2 months of cholesterol-enriched diet, despite increased serum cholesterol levels, no atheromatous plaque developed on the aortic wall. Impaired vascular function and reductions in the response to acetylcholine were related to changed endothelial cell function. Administration of a high cholesterol diet to YOS rat may represent a new model of mixed endogenous and exogenous hyperlipidemia that can resemble many human dislipidemic diseases and therefore may become a useful tool for the study of isolated endothelial dysfunction.

Acetylcholine↗
PubMed1994

Reduced smooth muscle cell regeneration in Yoshida (YOS) spontaneously hypercholesterolemic rats.

Hypercholesterolemia is a predisposing factor for atherosclerosis. We studied the response to damage of vascular smooth muscle cells (SMC) from normocholesterolemic Brown Norway (BN) and from spontaneously hyper-cholesterolemic Yoshida (YOS) rats (16-24 month old). The regrowth rate of SMC from BN and YOS rats after freeze-induced damage was similar in the presence of fetal calf serum and of serum derived from normocholesterolemic rats, while it was reduced in the presence of serum from hypercholesterolemic rats. Freeze-injury of the abdominal aorta was followed by reduced neointima formation in YOS rats, as compared to BN rats, confirming the impaired response of vascular cells from hypercholesterolemic rats to injury. This defect may be due either to lipids or to unknown factors present in the hyperlypidemic serum.

Animals↗
PubMed1994

Regeneration of the endothelium in the canine and feline thoracic duct.

Recent improvements in microsurgical techniques on lymphatic vessels facilitated the treatment of lymphoedema. But it is becoming clear that a successful treatment of lymphatic disease has to be based on knowledge of ongoing processes. Particularly, one of the important and unclear issue is cellular mechanisms of lymphatic regeneration. The regeneration of endothelial and the smooth muscle cells of the thoracic duct has been experimentally tested in vivo. The canine and feline thoracic duct was cryo-injured using 3 mm-based copper rod. Damaged endothelial cells remained attached to the substrate and lost unthrombogenecy within 48 hr. Adjacent EC restored the defect within 3 days by migration and proliferation. We observed that on the first day, the endothelial monolayer included some elongated multinuclear cells with blind silver lines whereas, on the third day, they were replaced by a population of smaller ECs with numerous mitoses. Organization of the monolayer was restituted within 7 days. The newly formed endothelium was similar to regenerating endothelium of arteries. In general, the clot that appeared at the zone of injury on the second day was dissolved by the third day. Occasionally, the dense polymorphic clot adhered to the wall and caused a delay in reendothelisation. Complete restitution of the tunica media which involved migration and proliferation mechanisms accompanied the endothelium regeneration.

Animals↗