Biomedical subjects

S Grimaldi

Publications and source records attributed to S Grimaldi.

At least 37 records · Page 2Linked to original sources

PubMed1993

Use of octadecylrhodamine fluorescence dequenching to study vesicular stomatitis virus fusion with human aged red blood cells.

Human erythrocytes were separated into five fractions representing different age groups. In each group phospholipid inside-outside translocation was determined by quantitation of the amino phospholipids phosphatidylserine and phosphatidylethanolamine and their lyso-derivatives by thin layer chromatography. To assess the role of transbilayer phospholipid distribution in the recognition and fusion of vesicular stomatitis virus (VSV) and human aged erythrocytes, we monitored the fusion kinetics using the octadecylrhodamine dequenching assay. Fusion of VSV with each single group of red blood cells (RBC) was not detectable with the youngest cells (F1 group) but increased with RBC aging (F2-F5 groups). The same increase in fusion was observed with microvesicles generated from RBC in which aging was mimicked by incubating the cells with Ca2+ in the presence of the Ca2+ ionophore A23187. Conversion of the aminophospholipids to the trinitrophenyl derivative by reaction with trinitrobenzensulfonate completely inhibits fusion on ghosts in which aging was artificially induced by translocation of aminophospholipids in the outer leaflet (symmetric ghosts). These results indicate that RBC become susceptible to VSV fusion during aging and in all pathology related to the aging process.

Erythrocyte Aging↗

PubMed1992

Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein.

Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.

Animals↗

PubMed1992

Charge and pH effect on the early events of Epstein-Barr virus fusion with lymphoblastoid cells (Raji).

Fusion of Epstein-Barr virus (EBV) with Raji cells was measured after exposure of the virus to neutral or low pH, enzymatic modification of the viral spike glycoproteins, or chemical modification of the target membrane. The relief of octadecylrhodamine (R18) fluorescence self-quenching was used to monitor fusion. Fusion of EBV with Raji cells at pH 5.9 was significantly enhanced compared to that at neutral pH. Treatment of Raji cells with agents known to modify the surface net charge (trinitrobenzene sulfonic acid) totally prevented fusion at a neutral pH. Desialylation of EBV significantly reduced the extent of fusion with Raji cells. Our results demonstrate that EBV is rapidly internalized and then fuses with lymphoblastoid cells in the endocytic vesicles.

Cells, Cultured↗

PubMed1990

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus.

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.

Electron Spin Resonance Spectroscopy↗

PubMed1990

Growth delay in Down syndrome and zinc sulphate supplementation.

Children affected with Down syndrome (DS) show deficient growth, immunodeficiency--especially concerning the T-cell population--and low plasma zinc levels. New growth charts have been recently proposed, and zinc supplementation to the diet has been reported to improve transiently the efficiency of the immune system. The aim of this study was to evaluate if in DS children zinc sulphate therapy could improve the growth rate and affect some endocrine parameters. We studied 22 patients (16 males and 6 females) who received zinc sulphate for 6 to 9 months. Fifteen of 22 patients studied reached a higher centile in their growth rate, whereas the remaining seven showed no change, at least to date. The average height velocity changed from 23.84 +/- 7.98 mm/6 months to 40.80 +/- 7.68 mm/6 months. Growth hormone serum level was 5.94 +/- 4.89 ng/ml compared with 7.49 +/- 6.75 ng/ml before and after therapy, respectively. Somatomedin serum level was 160.27 +/- 68.88 mU/ml and 205 +/- 124.07 mU/ml before and after therapy, respectively. In conclusion, zinc sulphate therapy of patients with DS affects not only the immune system, as previously reported, but can also accelerate growth.

Adolescent↗

PubMed1990

Hypothyroidism in patients with thalassemia syndromes.

Sixty transfusion-dependent thalassemic patients were studied by simultaneous measurement of circulating thyroid hormones, basal thyroid-stimulating hormone (TSH) and TSH response to thyrotropin-releasing hormone with the aim of evaluating the frequency of hypothyroidism in such patients, and the relationship between hypothyroidism and compliance with treatment and iron overload. Thyroid failure was present in 31 of the 60 patients. A correlation was found between impairment of thyroid functions, duration of chronic hypoxia and the activities of various transaminases. The results of this study emphasize the importance of early evaluation of thyroid function in thalassemic patients and suggest that anemia and hypoxia may potentiate the toxicity of iron deposition in endocrine glands.

Adolescent↗

PubMed1988

Enzymatic deglycosylation of human thyroglobulin: fluorescence studies.

The interaction between the carbohydrate and the amino acid residues in human thyroglobulin has been studied. Previous reports showed that the removal of the two terminal carbohydrates of the complex chains leads to an increase in thyroglobulin binding to thyroid membranes. In our study, after enzymatic release with glycosidases of the sugar moieties from thyroglobulin, a time-dependent decrease in tryptophan fluorescence has been observed. This decrease was also associated with a shift in the emission peak from 335 to 340 nm. The strong quenching of tryptophan emission was also accompanied by a decrease in the exposure of tryptophan residues, as shown by a Stern-Volmer analysis with the neutral quencher acrylamide. These data, together with the increase in fluorescence of the dansylated deglycosylated thyroglobulin, strongly suggest that a significant conformational change of thyroglobulin follows the deglycosylation of the protein.

Apoproteins↗

PubMed1988

Effect of ouabain binding on the fluorescent properties of the Na+/K+-ATPase.

The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.

Acrylamide↗

PubMed1987

TPA induction of Epstein-Barr virus early antigens in Raji cells is blocked by selective protein kinase-C inhibitors.

We have shown that modulation of intracellular calcium in EBV latently infected cells could induce the expression of viral antigens, and suggested that a protein kinase-C (PKC) may play a major role in the EBV genome activation. We now report further investigations on the role of PKC using 2 selective enzymatic inhibitors [1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine] (H-7) and Staurosporine. We show that these inhibitors can abrogate the inductive effect of TPA or the combination of TPA plus n-butyrate. The inhibitors have no effect on induction by calcium ionophores or by viral superinfection. In this context the effect of verapamil (a specific calcium channel blocker) and of several calmodulin antagonists was investigated. No inhibitory effect of these agents could be demonstrated on any of the induction systems examined. These observations strengthen the idea that in some instances cellular PKC plays a role in the expression of viral antigens; however, alternative regulatory mechanisms cannot be excluded.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1987

[Fluorescent properties of (Na+/K+)ATPase].

1. The effect of ouabain on the molecular properties of (Na+/K+)-ATPase has been studied in purified preparations of the enzyme, isolated from the microsomal fraction of outer red medulla of porcine kidney, according to a modification of the method described by Jorgensen. 2. Ouabain, a specific inhibitor of (Na+/K+)-ATPase, binds at the potassium site of the enzyme, thus generating an increase in its stability towards the common denaturing agents, such as exposure to different concentration of guanidinium chloride (GdmC1) or to acidic solutions.

Animals↗

PubMed1986

Calcium modulation activates Epstein-Barr virus genome in latently infected cells.

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.

Aminoquinolines↗

PubMed1986

Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma.

T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein). The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4). The 27K protein does not appear to be a fragment of TBG since 1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and 2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings: 1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4; 2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and 3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4. The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin. This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG. In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG. The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer. These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.

Antibodies, Monoclonal↗

PubMed1986

Biosynthesis of a novel thyroxine-binding protein (27K protein) in human hepatoma (Hep G2) cells.

Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4-binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein.

Animals↗

PubMed1986

[Effects of enzymatic deglycosylation of human goiter thyroglobulin on its immunochemical properties].

Thyroglobulin (Tg), isolated from soluble iodoproteins by ammonium sulphate fractionation, was enzymatically deglycosylated in vitro and analyzed by polyacrylamide gel electrophoresis, double immunodiffusion and non-commercial RIA. Carbohydrate and iodine content was chemically determined. By PAAGE deglycosylated Tg (dTg) showed the appearance of a major band in the 12S region and three slower migrating bands corresponding to higher aggregates than 19S Tg. In immunodiffusion by testing native and deglycosylated Tg against anti-native Tg antiserum it was shown the appearance of a spur of native on deglycosylated Tg. By RIA of native and deglycosylated Tg against anti-deglycosylated Tg antiserum it was shown a minor binding capacity of the anti-deglycosylated antibody against native Tg at high dilutions. The results demonstrate that the enzymatic deglycosylation release almost all the carbohydrates of goiter Tg and that the removal of the carbohydrates of Tg produces a loss of antigenic determinants of the molecule.

Electrophoresis, Polyacrylamide Gel↗

PubMed1986

[Polymorphism of human thyroxine binding serum globulin (TBG); existence of a new form of serum protein without detectable triiodothyronine binding power].

Two kinds of TBG polymorphism are described in human, one found in deglycosylated TBG from individual blood donors, the other is a genetically determined polymorphism. TBG from plasma samples from a patient with toxic goiter, not autoimmune, (p)TBG, from the patient's mother (m)TBG and from individual donors (n)TBG, were labeled with [125I]T4 or [125I]T3 and submitted to isoelectric focusing (IEF), followed by autoradiography. Three faint [125I]T4 radiolabeled bands were detectable in (p)TBG while four strong [125I]T4 radiolabeled bands were detectable in (m)TBG and (n)TBG), respectively. IEF of the [125I]T3 incubated serum samples resulted in no detectable isoelectric radiolabeled band for (p)TBG while a normal pattern was found in (m)TBG and in (n)TBG, respectively. These data suggest a new intraindividual not linked to sexual chromosome X polymorphism characterized by a loss in hormone binding.

Adult↗

PubMed1985

Interaction of carbohydrate and protein in thyroxine binding globulin.

The fluorescence properties of human thyroxine binding globulin were evaluated during enzymatic deglycosylation by using both neuraminidase and a mixture of glycosidases. Three fluorescent chromophores, one intrinsic and two extrinsic, were monitored, and all showed changes in fluorescent parameters that have been interpreted in terms of a loss of interactions between the carbohydrate and amino acid residues during deglycosylation. The loss of carbohydrates also results in a decrease in stability of the protein to both acid and guanidinium chloride inactivation. Since deglycosylation decreases the frictional ratio of thyroxine binding globulin, it is concluded that, although sialic acid and other sugar residues are in contact with the protein surface, the hydrated carbohydrate chains protrude partially into the solvent.

Acrylamide↗

PubMed1983

Polymorphism of human thyroxine-binding globulin.

T4-binding globulin (TBG) consists of a single polypeptide chain containing 4 oligosaccharide units with an average of 10 terminal sialic acid residues. Isoelectric focusing (IEF) of TBG purified from pooled human plasma showed 4 main bands isoelectric at pH 4.2, 4.3, 4.5, and 4.6. Removal of 85% of carbohydrates by treatment with mixed glycosidases reduced these bands to 2, at pH 5.4 and 5.7. This residual microheterogeneity was not related to protein-ampholyte complexes, since it was still present in 8 M urea. It also did not represent an equilibrium mixture of interchangeable conformations, since each band obtained in the first dimension IEF gave a single spot when rerun in a second dimension. Therefore, the residual microheterogeneity of TBG after removal of carbohydrates can be attributed to variation in amino acid composition. Since genetic polymorphism of TBG was recently demonstrated, we further investigated whether the residual microheterogeneity was genetically determined. Plasma samples from 20 white donors and 17 black donors were labeled with [125I]T4 and submitted to IEF, followed by autoradiography. TBG-1, found in white donors and most black donors, showed the same 4 bands as TBG purified from pooled plasma. Two less frequent phenotypes were found in black individuals: TBG-2, with 4 bands at approximately pH 4.25, 4.45, 4.55, and 4.7; and TBG-1,2, having all of the bands present in TBG-1 and TBG-2. Electrophoretically homogeneous preparations of TBG of each type were obtained from 100 ml plasma; after deglycosylation, TBG-1 revealed 2 bands isoelectric at pH 5.4 and 5.7, TBG-2 had 2 bands at pH 5.7 and 5.9, and TBG-1,2 had 3 bands at pH 5.4, 5.7, and 5.9. The same TBG bands were found after neuraminidase treatment of whole plasma from the same donors. These data demonstrate two kinds of TBG polymorphism. The first is found in deglycosylated TBG from individual donors and is probably due to variation in amino acid composition. The second, also unrelated to the carbohydrate moiety, is a genetic polymorphism found in blacks.

Black People↗

PubMed1982

The isoelectric focusing of human thyroglobulin.

The isoelectric focusing of human thyroglobulin has been studied on slab gels. Three bands, focusing between pH 4.4 and 4.7, are observed. Deglycosylation of thyroglobulin does not affect the distribution of focused bands, but increases the pH range of focusing slightly. Native thyroglobulin and its half-sized subunit show the same distribution of isoelectric bands. Refocusing of one band results in the appearance of the three original bands. It appears that soluble complexes of thyroglobulin with ampholyte account for the apparent heterogeneity observed on isoelectric focusing.

Ampholyte Mixtures↗