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Biomedical subjects

M Levi

Publications and source records attributed to M Levi.

At least 199 records · Page 11Linked to original sources
PubMed1997

Flow cytometry, sorting and immunocharacterization with proliferating cell nuclear antigen of cycling and non-cycling cells in synchronized pea root tips.

In the 3-d-old 2-mm root tip of Pisum sativum L. cv. Lincoln the percentage of actively proliferating cells is estimated to be 70%. The remaining cells are non-cycling and arrested with 2C and 4C DNA content in G0 and in G2Q, respectively. In this work we studied the kinetic significance of these quiescent cells, using the sorting capabilities of flow cytometry and immunofluorescence techniques to detect the proliferation marker PCNA (proliferating cell nuclear antigen) inside cells within the different cell-cycle compartments. While in animal cells, PCNA is present at a high level only in actively proliferating cells, in 3-d-old pea root tips 95% of the cells are PCNA-positive. After flow cytometry and sorting of pea non-cycling nuclear populations, all G2Q nuclei appeared strongly PCNA-positive, indicating that these cells had recently left the cell cycle. By contrast, most G0 nuclei showed a low level of PCNA immunofluorescence intensity, as measured by image analysis, with about 25% of the nuclei being PCNA-negative. This small percentage was found to correspond to root cap cells, as could be observed in the root tip section. These are the only cells in the root apical region which are fully differentiated and which, therefore, lack the competence to enter the cell cycle. In contrast, the more or less PCNA-positive G0 nuclei could represent a kinetically heterogeneous population of cells competent to proliferate, but which have either recently left the cell cycle or are progressing to the G0-G1 transition.

Cell Cycle↗
PubMed1997

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes.

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.

2-Naphthylamine↗
PubMed1997

In K562 and HL60 cells membrane ageing during cell growth is associated with changes in cholesterol concentration.

In a cell culture model of aging we have previously shown that there is an age-related decrease in the lipid dynamics of the proerythropoetic K562 cell membranes, as determined by the generalized polarization (GP) of the phase-sensitive lipid probe 2-dimethylamino-6-lauroylnaphthalene (Laurdan) (T. Parasassi, M. Di Stefano, G. Ravagnan, O. Sapora and E. Gratton. Exp. Cell Res., 202 (1992) 432-439). In the present study we also extended our observations to the lymphoblastoid HL60 cell line. In both K562 and HL60 cells during the four days after the last cell culture medium renewal the GP Laurdan value increased in a linear fashion indicating a time-dependent decrease in lipid dynamics. The initial membrane physical properties were almost completely restored upon renewal of the cell culture medium. We measured lipid composition, including individual and total phospholipids, free and esterified cholesterol at the first ('young') and at the fourth ('aged') day after culture medium renewal. We found that the decreased membrane lipid 'fluidity' at the fourth day of cell growth was associated with a 40% increase in cholesterol concentration in both cell lines. This increase in cholesterol concentration was reversible 24 h following the culture medium change. We conclude that in K562 and HL60 cells the 'age-related' decrease in membrane lipid dynamics is mediated by an 'age-related' increase in cell cholesterol content.

2-Naphthylamine↗
PubMed1997

Pharmacokinetics and antiepileptic activity of valproyl hydroxamic acid derivatives.

PURPOSE: To explore the utilization of seven novel hydroxamic acid derivatives of valproic acid (VPA) as new antiepileptics. METHODS: The study was carried out by investigating the pharmacokinetics of two active compounds in dogs and pharmacodynamics (anticonvulsant activity and neurotoxicity) of valproyl hydroxamic acid and six of its derivatives. RESULTS: Three valproyl hydroxamic acid derivatives: valproyl hydroxamic acid-VPA-HA, N-(1-hydroxyethyl)-valpromide-HEV and N-methoxy valpromide, showed better anticonvulsant activity than VPA at the maximal electroshock (MES) test. The remaining four compounds, O-valproyl-VPA-HA, N-valproyl-O-valproyl-VPA-HA, N-(1-methoxyethyl) valpromide and N-(1,2-dihydroxylpropyl)-valpromide were found to be inactive. Therefore, only the pharmacokinetics of the active compounds VPA-HA and HEV was studied. CONCLUSIONS: In contrast to valpromide (VPD) which is biotransformed to VPA, VPA-HA and HEV were found to be stable in vivo to the biotransformation of the amide to its corresponding acid. VPA-HA and HEV showed improved anticonvulsant activity over VPA because of their greater intrinsic activity and not due to better pharmacokinetic characteristics. This paper discusses the structural requirements for active anticonvulsant valproyl hydroxamic acid derivatives.

Animals↗
PubMed1997

Linear epitope mapping of humoral responses induced by vaccination with recombinant HIV-1 envelope protein gp160.

To enhance utility of the linear epitope mapping (Pepscan) technique for assay of humoral responses linked to vaccination, two modifications were tested. First, peptides were incubated with serum contained in baths rather than individual wells. Second, a rigorous statistical model was developed to determine which peptide/antibody-binding interactions were significant. The modifications increased the ability to detect signal in these experiments by 15- to 45-fold. These two modifications were applied to linear epitope mapping of HIV seropositive volunteers under treatment with recombinant HIV gp160 and also to rabbits immunized with the same product. Changes in fine specificity of response were observed in animal models and human vaccine recipients over the course of an immunization series with this antigen.

AIDS Vaccines↗
PubMed1997

Coagulation abnormalities in type 1 Gaucher disease are due to low-grade activation and can be partly restored by enzyme supplementation therapy.

In type 1 Gaucher disease a bleeding tendency occurs which is partly caused by thrombocytopenia due to massive splenomegaly. In addition, low levels of factors IX and XI have been described. The mechanism responsible for these clotting factor abnormalities is unknown. We performed a detailed study of parameters of coagulation and fibrinolysis in 30 type 1 Gaucher disease patients (14 splenectomized) before and after treatment with enzyme supplementation therapy. Pre-treatment aPTT and PT were prolonged in 42% and 38% of patients, respectively. In 30-60% serious deficiencies (< 50%) of coagulation factors XI, XII, VII, X, V and II were observed. The low levels of factor V correlated with platelet count and were inversely associated with splenic volume. Levels of inhibitors were mildly decreased. Markers for activation of coagulation (thrombin-antithrombin (TAT) complex) and fibrinolysis (PAP complex, fibrin cleavage product D-dimer) were significantly elevated, especially in the splenectomized patients, indicating ongoing activation of these processes. After 12 months of enzyme supplementation therapy partial correction occurred. Thus, severe disorders of the coagulation system occur in Gaucher disease, contributing to the bleeding tendency. The deficiencies may be the result of consumption of coagulation factors caused by ongoing low-level coagulation activation. possibly due to mononuclear cell activation.

Adult↗
PubMed1997

The cytokine-mediated imbalance between coagulant and anticoagulant mechanisms in sepsis and endotoxaemia.

Disseminated intravascular coagulation (DIC) is a frequently occurring complication of sepsis and may contribute to multiple organ failure. More insight into the pathogenesis of this derangement of the coagulation system is necessary to develop more effective therapeutic strategies for this condition. Recently, more detailed knowledge on the pathogenetic pathways involved in DIC has been obtained by the study of models of experimental bacteraemia and endotoxaemia in human subjects and non-human primates. The mechanisms that lead to activation of coagulation, potentiated by the simultaneous depression of physiological inhibitory systems and to impaired function of the fibrinolytic system, are outlined in this review. In addition, the mediatory role of various cytokines in the derangement of coagulation is discussed.

Anticoagulants↗
PubMed1997

Effect of glucocorticoids on neonatal rabbit renal cortical sodium-inorganic phosphate messenger RNA and protein abundance.

Administration of glucocorticoids to neonates increases proximal tubule volume absorption by increasing glucose, bicarbonate, and amino acid transport. We have recently demonstrated that glucocorticoids may contribute to the maturational decrease in phosphate transport. This study examines the maturation of NaPi-6 [the regulated proximal tubule sodium-inorganic phosphate (Na-Pi) transporter] mRNA and protein abundance and the mechanism for the decrease in phosphate transport by glucocorticoids. Weaned young rabbits (5 wk) had a 2-fold greater brush border membrane NaPi-6 protein abundance than that measured in adults. Renal cortical NaPi-6 mRNA abundance was comparable in neonates (less than 10 d of age) and adults. Renal brush border membrane vesicles from dexamethasone-treated neonatal rabbits (10 micrograms/100 g of body weight for 4 d) had a lower rate of Na-Pi transport than vehicle-treated controls (46.8 +/- 6.5 versus 71.0 +/- 9.0 pmol 32P/10 s/mg of protein, p < 0.05). Abundance of NaPi-6 protein in brush border membrane vesicles was 3-fold lower in newborn rabbits treated with pharmacologic doses of dexamethasone than in vehicle-treated controls. NaPi-6 mRNA abundance was the same in both groups. NaPi-1, a brush border membrane phosphate transporter which is also an anion channel, mRNA, and protein abundance was not affected by glucocorticoids. These data demonstrate that there is a maturational decrease in NaPi-6 protein abundance and that glucocorticoids decrease neonatal phosphate transport, at least in part, by reducing the number of Na-Pi transporters.

Animals↗
PubMed1997

Differential effects of reconstituted high-density lipoprotein on coagulation, fibrinolysis and platelet activation during human endotoxemia.

High-density lipoproteins (HDL) can bind and neutralize lipopolysaccharides (LPS) in vitro and in vivo. HDL can also affect fibrinolytic activity and can directly influence platelet function by reducing platelet aggregation. In this study, the effects of reconstituted HDL (rHDL) on LPS-induced coagulation, fibrinolysis and platelet activation in humans were investigated. In a double-blind, randomized, placebo-controlled, cross-over study, eight healthy male volunteers were injected with LPS (4 ng/kg) on two occasions, once in conjunction with rHDL (40 mg/kg, given as a 4 h infusion starting 3.5 h prior to LPS injection), and once in conjunction with placebo. rHDL significantly reduced LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2) and fibrinolysis (plasma levels of tissue type plasminogen activator antigen, t-PA). No effect was observed on LPS-induced inhibition of the fibrinolytic pathway (PAI-1) or on the transient thrombocytopenia elicited by LPS. Furthermore, rHDL treatment significantly enhanced the inhibition of collagen-stimulated inhibition of platelet aggregation during endotoxemia, but had no such effect on arachidonate-stimulated platelet aggregation. rHDL treatment per se also reduced collagen-induced platelet aggregation. These results indicate that rHDL modifies the procoagulant state associated with endotoxemia.

Adult↗
PubMed1996

Release of bactericidal/permeability-increasing protein in experimental endotoxemia and clinical sepsis. Role of tumor necrosis factor.

Bactericidal/permeability-increasing protein (BP]) is contained within the azurophilic granules of neutrophils and is able to neutralize endotoxin and kill Gram-negative bacteria. TNF has been implicated as a mediator of endotoxin-induced neutrophil degranulation. To assess the role of TNF in the elevated BPI levels during sepsis, the following studies were performed. 1) In 31 consecutive patients with sepsis syndrome, plasma BPI levels were markedly elevated compared with those in healthy controls, but showed no correlation with simultaneously measured TNF concentrations. 2) In four healthy men, i.v. injection of recombinant human TNF (50 microg/m2) induced a rapid rise in plasma BPI levels. 3) In eight normal subjects, i.v. administration of Escherichia coli endotoxin (4 ng/kg) elicited subsequent increases in the plasma concentrations of TNF and BPI. 4) Eight healthy chimpanzees were investigated after i.v. injection of endotoxin (4 ng/kg); four animals received endotoxin only, and four animals received an anti-TNF mAb simultaneously. Although anti-TNF completely prevented the endotoxin-induced appearance of TNF activity, the rise in BPI levels remained unaltered. These results suggest that TNF is not critical for the release of BPI from neutrophils during experimental endotoxemia or clinical sepsis.

Adult↗
PubMed1996

Immunodominant glycoprotein 41 epitope identified by seroreactivity in HIV type 1-infected individuals.

A highly conserved gp41 epitope (amino acid sequence ELDKWA) has been described to elicit antibodies neutralizing a broad variety of HIV-1 strains. We analyzed the antibody reactivity of HIV-1-infected individuals from Argentina and Sweden to overlapping synthetic peptides covering aa647-684 of HIV-1 MN gp41. An immunodominant antigenic determinant was discovered in the C-terminal region adjacent to the ELDKWA sequence. Most of the sera from both Argentina and Sweden reacted only with peptides representing this region. Two other patterns of reactivity were also observed: some sera reacted only with peptides spanning the central region of gp41, including the ELDKWA sequence; other sera reacted with both the central and C-terminal regions. Differences in reactivity were noted between Argentinian and Swedish sera in terms of peptides covering the central region. In addition, to determine the amino acids essential for antibody binding, seroreactivity against a set of substitution peptides covering the immunodominant epitope was studied. Results obtained indicated that carboxyl amino acids WNWFDI close to the ELDKWA sequence were the most important for antibody binding. Ability of sera, tested for peptide reactivity, to neutralize HIV-1 was also analyzed. High antibody reactivity to the central region was frequently found in sera with high neutralizing titers. This observation was not seen when seroreactivity to peptides spanning the C-terminal region was analyzed. These results suggest that the immunodominant epitope C terminal to the ELDKWA sequence is not involved in inducing neutralizing antibodies.

Amino Acid Sequence↗
PubMed1996

Comparison of sustained antithrombotic effects of inhibitors of thrombin and factor Xa in experimental thrombosis.

BACKGROUND: In the pathogenesis of (recurrent) thrombosis, clot-associated thrombin appears to play an important role. Antithrombin III-independent thrombin inhibitors have been shown to neutralize clot-bound thrombin effectively. We compared the sustained antithrombotic effects and the effects on endogenous fibrinolysis of several of these agents with recombinant tick anticoagulant peptide (rTAP), a selective factor Xa inhibitor, and low-molecular-weight heparin (LMWH) in an experimental venous thrombosis model. METHODS AND RESULTS: Rabbits received either recombinant hirudin (rHir), Hirulog-1, CVS#995 (a novel direct inhibitor of thrombin), rTAP, LMWH, or saline. The effect on thrombus growth was assessed by measuring the accretion of 125I-labeled fibrinogen onto preformed nonradioactive thrombi, and the effect on endogenous fibrinolysis was assessed by measuring the decline in radioactivity of preformed 125I-labeled thrombi in rabbit jugular veins. All direct thrombin inhibitors induced a sustained antithrombotic effect compared with either LMWH and rTAP. In addition, CVS#995 also further decreased thrombus size after stopping its infusion, which was due to a significant enhancement of endogenous fibrinolysis. CONCLUSIONS: Direct thrombin inhibition by rHir, Hirulog-1, or CVS #995 induces a sustained antithrombotic effect compared with rTAP and LMWH, which is most likely due to inhibition of clot-bound thrombin. CVS#995 was shown to also enhance the extent of endogenous fibrinolysis to a greater degree compared with rHir and might therefore be an interesting new antithrombotic agent for the treatment of venous and arterial thrombosis.

Animals↗
PubMed1996

Adrenal suppression among asthmatic children receiving chronic therapy with inhaled corticosteroid with and without spacer device.

BACKGROUND: Inhaled corticosteroids have become a first-line treatment for chronic asthma. It has been shown that inhaled corticosteroids can have a measurable effect on the hypothalamic-pituitary-adrenal axis in asthmatic children. OBJECTIVE: To investigate the prevalence of adrenal suppression among asthmatic children receiving chronic therapy with low to moderate doses (up to 1000 micrograms) of inhaled beclomethasone dipropionate via a metered dose inhaler (MDI) and via MDI attached to a spacer device (MDI-spacer). METHODS: The study included 39 asthmatic children currently undergoing therapy; 24 received beclomethasone dipropionate by MDI attached to a spacer, and 15 directly by MDI. All the patients had been treated for at least 4 months. Another 21 children were normal controls. The 24-hour urinary free cortisol excretion was measured to evaluate hypothalamic-pituitary-adrenal axis function. RESULTS: Seven of 15 (47%) patients from the MDI group had reduced 24 hour-urinary free cortisol excretion and 2 of 24 (8%) in the MDI-spacer group (P = .006). The mean 24-hour urinary free cortisol excretion of the MDI group was 0.0185 +/- 0.0089 microgram/gram creatinine, and the MDI-spacer and the control groups were, 0.0290 +/- 0.0138 microgram/gram creatinine and 0.0270 +/- 0.0118 microgram/gram creatinine, respectively, (P = 0.37, f = 3.51 ANOVA). CONCLUSION: Chronic inhalation of low to moderate doses of corticosteroids is associated with adrenal suppression in some asthmatic children. This side effect is more common among patients inhaling directly from the MDI and is less frequent when a large volume spacer is attached to the MDI.

Administration, Inhalation↗