Biomedical subjects

R Donato

Publications and source records attributed to R Donato.

At least 55 records · Page 3Linked to original sources

PubMed1995

S-100 protein and annexin II2-p11(2) (calpactin I) act in concert to regulate the state of assembly of GFAP intermediate filaments.

S-100 protein and annexin II2-p11(2) were reported to inhibit and to stimulate the assembly of glial fibrillary acidic protein (GFAP), respectively, in a Ca(2+)-dependent manner. Here we show by a number of experimental approaches that S-100 protein contrasts all the effects of annexin II2-p11(2) on GFAP assembly and, conversely, that annexin II2-p11(2) contrasts the inhibitory effects of S-100 protein on GFAP assembly, in a dose-dependent manner in both cases. Altogether, these data suggest that two specific Ca2+ effectors, i.e., annexin II2-p11(2) and S-100 protein, might regulate the state of assembly of glial filaments in a concerted manner.

Animals↗

PubMed1995

Membrane-bound guanylate cyclase as the receptor of natriuretic peptides. A minireview.

A minireview is presented on the ultracytochemical localization of membrane-bound guanylate cyclase (GC) in various tissues and in cultured cells after activation with three natriuretic peptides, the atrial natriuretic factor (ANF), the brain natriuretic peptide (BNP), and the C-type natriuretic peptide (CNP). GC, two subtypes of which have been recently identified, is the receptor for these peptides. The GC isoforms are differently stimulated by ANF, BNP and CNP. Under our experimental conditions, the natriuretic peptides were strong activators of GC since samples incubated without natriuretic peptides do not reveal any cyclase activity. The natriuretic peptide-stimulated GC activity was studied in rat kidney, lung, adrenal gland and neurohypophysis, in rabbit platelets, in lamb olfactory mucosa, and in rat C6 glioma cells. On the basis of the subcellular GC localization some additional functions of peptides are hypothesized.

Animals↗

PubMed1995

Role of leukaemia inhibitory factor (LIF) in rat peripheral nerve regeneration.

The cytokine leukaemia inhibitory factor (LIF) favours the survival and growth of axons in vitro. The efficacy of this factor in the in vivo model has been the aim of this study. Following nerve transection and immediate entubulation repair in the rat sciatic nerve, this study demonstrated that (1) LIF promotes the growth of a population of axons of greater cross-sectional area after 6 and 12 weeks in comparison to either saline (negative control) or basic fibroblast growth factor (bFGF) (positive control), (2) LIF improves the nerve conduction velocity of regenerating axons, (3) LIF has a positive effect on skeletal muscle mass following nerve repair, (4) the benefits of LIF on skeletal muscle appear to be somewhat independent of reinnervation as similar observations are made where there is no growth of a tissue bridge within the tube, and (5) the effects of LIF seem to be potentiated by the addition of fibronectin.

Animals↗

PubMed1995

Annexins in the gastropod nervous system.

We studied immunologically the distribution of the phospholipid- and Ca(2+)-binding proteins annexin I and annexin V in the nervous system of the molluscs, Aplysia californica and Helix pomatia. Within the Aplysia central nervous system we identified annexin I-immunoreactive neurons in the cerebral, buccal and abdominal ganglia. Annexin V-like material is restricted to neurons in the pedal ganglia. The Helix central nervous system contains annexin I-immunoreactive neurons in cerebral, buccal, visceral, left and right parietal, and pedal ganglia. Annexin V-immunoreactive material is present in neurons of the pleural, left and right parietal, and visceral ganglia.

Animals↗

PubMed1994

Mechanism of S100 protein-dependent inhibition of glial fibrillary acidic protein (GFAP) polymerization.

S100 protein, a subfamily of Ca(2+)-binding proteins of the EF-hand type, was recently shown to bind to and to inhibit the polymerization of the glial fibrillary acidic protein (GFAP), the intermediate filament component of astroglial cells, in the presence of micromolar levels of Ca2+ (J. Biol. Chem. 268, 12669-12674). By a sedimentation assay and viscometry we show here that S100 protein interferes with the very early steps of GFAP polymerization (nucleation) and with the GFAP polymer growth, thereby retarding the onset of GFAP assembly, reducing the rate and the extent of GFAP assembly, and increasing the critical concentration of GFAP assembly. Moreover, S100 protein disassembles preformed glial filaments. All the above effects can be explained by sequestration of soluble GFAP by S100 protein, as also indicated by the stoichiometry of S100 protein binding to GFAP and of S100 protein effects on GFAP assembly. Our data suggest that S100 protein might serve the function of avoiding excess GFAP polymerization and might participate in remodeling of glial filaments following elevation of the intracellular free Ca2+ concentration. Also, our data lend support to the notion that intermediate filaments are dynamic cytoskeleton structures that assemble and disassemble, and to the existence of cytoplasmic factors implicated in the regulation of the state of assembly of intermediate filaments.

Animals↗

PubMed1994

Calpactin I binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments in a Ca(2+)-dependent manner: implications for concerted regulatory effects of calpactin I and S100 protein on glial filaments.

Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental conditions. Cosedimentation of calpactin I with GF, which occurs at micromolar free Ca2+ concentrations, is proportional to the concentrations of both calpactin I and GFAP and does not occur under conditions where GFAP assembly is maximally inhibited by, e.g., S100 protein. Annexin II also cosediments with GF and binds to GFAP, although to much smaller extents. Other annexins, such as annexins I, V, and VI, or p11 do not bind to either GF or GFAP. Calpactin I and S100 protein bind to different sites on GFAP, as investigated by fluorescence spectroscopy using acrylodan-labeled GFAP. Calpactin I and S100 protein might act, in the presence of Ca2+, in a concerted manner to determine the number and topography of GF in differentiating and/or mature glial cells.

2-Naphthylamine↗

PubMed1994

Annexin V as a probe of the contribution of anionic phospholipids to the procoagulant activity of tumour cell surfaces.

The ability of anionic phospholipids (especially phosphatidylserine, PS) on the outer membrane leaflet of four tumour cell lines to support different stages of the extrinsic pathway of coagulation was probed using annexin V as an inhibitor. The procoagulant activity of two tumorigenic (MKN-28, human gastric carcinoma, Hep3B, human hepatoblastoma) and two non-tumorigenic (HepG2, human hepatocellular, HOC-1, human ovarian carcinoma) cell lines were observed to be inhibited by annexin V, although significant differences (observed as IC50 with respect to annexin V) were noted for each stage of coagulation and between different cell types. This was considered to suggest a restricted accessibility of PS in the vicinity of coagulation factors on the surface of the cell. PS levels, as estimated by binding of 125I-annexin V, were high on two of the cell lines tested, equivalent to 24 x 10(6) sites per cell for HepG2 (Kd 128 nM) and 6.5 x 10(6) sites per cell for MKN-28 (Kd 50 nM). During 9 days' culturing of HepG2 and MKN-28, the number of sites per cell remained constant. However, perhaps supporting a proposal of reduced availability, there was an observed fall in PS-dependent procoagulant activity of HepG2 and MKN-28 cells, subsequent to a peak on reaching confluency at 3 days. Both prothrombinase activity and total procoagulant activity fell, even though the number of 125I-annexin V binding sites remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Anions↗

PubMed1993

Annexins V and VI in rat tissues during post-natal development: immunochemical measurements.

Annexins V and VI, two Ca(2+)-dependent phospholipid- and membrane-binding proteins, were immunochemically measured in a number of rat organs and tissues during post-natal development. Annexin V proved much more abundant than annexin VI irrespective of the organ and the post-natal period considered. In the brain, annexin V accumulated at a high rate from the end of the first post-natal week onward, whereas annexin VI was expressed in extremely low amounts irrespective of the period investigated. In contrast, the levels of both annexins in the heart were nearly constant, in the post-natal period investigated. In skeletal muscles, annexin V and VI levels were high around post-natal day 1 and decreased thereafter. A similar pattern was observed for annexin V in liver, whereas the amounts of annexin VI in this organ were at the limits of detectability. In the lung, annexin V accumulated almost linearly from birth to adulthood, whereas annexin VI was relatively high at birth, decreased to low levels by the end of the first post-natal week, and re-accumulated thereafter. Among the organs examined, the lung and heart proved the richest sources of annexins V and VI. Annexin V appears to be a useful marker of and to be implicated in brain, lung and skeletal muscle maturation.

Aging↗

PubMed1993

S-100 protein, but not calmodulin, binds to the glial fibrillary acidic protein and inhibits its polymerization in a Ca(2+)-dependent manner.

S-100 protein, a Ca(2+)-binding protein of the EF-hand type, interacts with the glial fibrillary acidic protein (GFAP) in a Ca(2+)-dependent manner. The binding of S-100 protein to GFAP was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and cross-linking experiments using the bifunctional cross-linker, disuccinimidyl suberate. The binding affinity was observed to be in the nanomolar range with a stoichiometry of 2 mol of GFAP/mol of S-100 protein (dimer). S-100 protein was found to inhibit the polymerization of GFAP in a dose- and Ca(2+)-dependent manner, with a half-maximal effect at an S-100 protein/GFAP molar ratio of 0.2 and maximal effect at a molar ratio of 0.5. Identical results were obtained irrespective of whether the unfractionated bovine brain S-100 protein mixture (S-100a plus S-100b), S-100ao, S-100a, or S-100b was used. S-100 protein was observed to be maximally effective as an inhibitor of GFAP polymerization at approximately 3 microM free Ca2+. Calmodulin neither bound to GFAP nor inhibited its polymerization. Altogether, the present results suggest that S-100 protein might be involved in the regulation of the state of assembly of glial filaments by binding to and sequestering unpolymerized GFAP.

2-Naphthylamine↗

PubMed1993

Immunocytochemical analyses of annexin V (CaBP33) in a human-derived glioma cell line. Expression of annexin V depends on cellular growth state.

The subcellular distribution of annexin V, a calcium-dependent phospholipid- and membrane-binding protein, in a human-derived cell line, GL15, was investigated by immunocytochemistry at light and electron microscope levels. Annexin V was found diffusely in the cytoplasm and associated with plasma membranes, membranes delimiting cytoplasmic vacuoles, membranes of the endoplasmic reticulum, and filamentous structures the identity of which remains to be established. By immunocytochemistry at the light microscope level and immunochemistry, the expression of annexin V in these cells was found to depend on cellular growth stage, being maximal soon after plating and progressively declining thereafter. However, re-expression of annexin V was observed whenever cell proliferation slowed down or arrested. These findings suggest that annexin V in glioma cells is mostly expressed in connection with cell differentiation. Also, the present ultrastructural data suggest that plasma membranes, membranes of the endoplasmic reticulum and the cytoskeleton are prominent sites of action of annexin V in vivo, thus lending support to the possibility that this protein might have a role in the regulation of cytoskeleton elements and/or of the structural organization of membranes.

Animals↗

PubMed1993

Time-resolved fluorescence of S-100a protein in the absence and presence of calcium and phospholipids.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca(2+)-binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca2+. Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca2+. Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca2+. Our data suggest that the conformation of the protein and its Ca(2+)-binding properties vary depending on the characteristics of charge and structure of phospholipids.

Animals↗

PubMed1993

Hospital acquired rota virus infection: the economics of prevention.

Nosocomial rota virus infection results in costs to patients (for example, delayed procedures) and to hospitals (for example, additional bed-days, and ward closures if an epidemic occurs). This paper analyses the economic efficiency of using a preventive measure--a high antibody milk supplement--in a hospital for children. The cost of implementing this program is compared with the benefits of avoiding nosocomial rota virus infection. The results of the study suggest that, taking a range of realistic values for the most significant variables, the use of the supplement would be cost-effective.

Animals↗

PubMed1993

Immunocytochemical localization of annexins V and VI in human placentae of different gestational ages.

The cellular and subcellular localization of annexins V and VI, two members of a superfamily of Ca(2+)-dependent phospholipid- and membrane-binding proteins, was investigated in chorionic villi of human placentae of different gestational ages by postembedding immunocytochemistry at the electron microscope level. All cell types of placental villi, i.e., the syncytiotrophoblast, Langhans cells, Hofbauer cells, fibroblasts, and capillary endothelial cells, appeared to express the two proteins, irrespective of the gestational age. By immunogold particle counts, annexin V was observed to be 2-3 times as much abundant as annexin VI. Syncytiotrophoblast cells appeared to contain the largest amounts and Langhans cells appeared to contain the least amounts of annexins V and VI, as judged by immunocytochemistry. The two proteins were found associated with plasma, Golgi, and vacuolar membranes, and with membranes of the endoplasmic reticulum, as well as diffusely in the cytoplasm. Annexin V appeared to be distributed in nearly equal proportions between cell membranes and the cytoplasm in stromal cells and to be about 30% associated with cell membranes in trophoblast cells, whereas annexin VI appeared almost equally distributed between cell membranes and the cytoplasm in trophoblast and stromal cells. Also, annexins V and VI appeared to be more abundant in trophoblast cells than in stromal cells. The present data strongly support the idea that placenta is a preferential site of annexin-regulated activities, and suggest that annexins V and VI are actively involved in the Ca(2+)-dependent regulation of membrane processes in trophoblast cells.

Annexin A5↗

PubMed1992

S-100 protein binds to annexin II and p11, the heavy and light chains of calpactin I.

S-100 protein, a dimeric, Ca(2+)-binding protein of the EF-hand type, interacts with annexin II (p36, the heavy chain of the cytoskeletal protein complex, calpactin I), with p11 (the light and regulatory chain of calpactin I) and with the hetero-tetramer annexin II2-p11(2) (calpactin I) in a Ca(2+)-regulated way, but not with annexins I, V and VI. The interaction of S-100 protein with the above proteins was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and acrylodan-annexin II and by cross-linking experiments using the bifunctional cross-linker disuccinimidyl suberate (DSS). S-100 protein binds with the highest affinity to annexin II (Kd approx. 0.4 microM) and with the lowest affinity to calpactin I (Kd approx. 10 microM), with a constant stoichiometry of about 2 mol of protein/S-100 dimer. Thus, S-100 protein could substitute for p11 in regulating the activities of annexin II in cells which do not express p11 and/or act synergistically with p11 in cells expressing both p11 and S-100. The binding of S-100 protein to p11 could reflect the natural tendency of S-100 subunits and p11 to dimerize. Chimeric p11-S-100 alpha and p11-S-100-beta proteins could therefore form in a Ca(2+)-regulated way. The interaction of S-100 protein with calpactin I appears of doubtful physiological importance, because of the low binding affinity, of the small extent of fluorescence changes induced by calpactin I in acrylodan-S-100 protein and of lack of DSS-induced complex formation between the two protein species.

2-Naphthylamine↗

PubMed1992

Novel isoforms of CaBP 33/37 (annexin V) from mammalian brain: structural and phosphorylation differences that suggest distinct biological roles.

Two calcium-dependent phospholipid- and membrane-binding proteins have been purified from bovine brain. These are termed CaBP33 and CaBP37. Complete sequence analysis has revealed that these two proteins are isoforms of annexin V. Despite an apparent difference of 4 kDa between the two proteins on SDS-PAGE, only two amino-acid substitutions were found. These are, in CaBP33, Ser-36 and Lys-125 and in CaBP37, Thr-36 and Glu-125. This corresponds to a mass difference of 15 Da. This was confirmed by electrospray mass spectrometric analysis. Both isoforms can be phosphorylated substoichiometrically in vitro by protein kinase C at residue Thr-22.

Amino Acid Sequence↗

PubMed1992

Membrane-bound annexin V isoforms (CaBP33 and CaBP37) and annexin VI in bovine tissues behave like integral membrane proteins.

The distribution of annexin V isoforms (CaBP33 and CaBP37) and of annexin VI in bovine lung, heart, and brain subfractions was investigated with special reference to the fractions of these proteins which are membrane-bound. In addition to EGTA-extractable pools of the above proteins, membranes from lung, heart, and brain contain EGTA-resistant annexins V and VI which can be solubilized with detergents (Triton X-100 or Triton X-114). A strong base like Na2CO3, which is usually effective in extracting membrane proteins, only partially solubilizes the membrane-bound, EGTA-resistant annexins analyzed here. Also, only 50-60% of the Triton X-114-soluble annexins partition in the aqueous phase, the remaining fractions being recovered in the detergent-rich phase. Altogether, these findings suggest that, by an as yet unknown mechanism, following Ca(2+)-dependent association of annexin V isoforms and annexin VI with membranes, substantial fractions of these proteins remain bound to membranes in a Ca(2+)-independent way and behave like integral membrane proteins. These results further support the possibility that the above annexins might play a role in membrane trafficking and/or in the regulation of the structural organization of membranes.

Animals↗

PubMed1992

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.

Animals↗

PubMed1992

Serum autoantibodies against glial fibrillary acidic protein in brain aging and senile dementias.

Autoantibodies against glial fibrillary acidic protein (GFAP) were investigated by ELISA test in sera of patients suffering from senile dementias and in healthy aging people. One hundred eight subjects divided into control, vascular dementia (VD), presenile Alzheimer's disease (AD), and senile Alzheimer's disease (SDAT) groups were included in the study. VD patients showed the highest antibody titers when compared to controls, whereas AD had the lowest titers when compared to the other groups. These results do not support the utility of anti-GFAP antibodies as useful markers of Alzheimer's disease, suggesting that their presence is a secondary phenomenon to blood-brain barrier disruption.

Aged↗