Biomedical subjects

D Frenkel

Publications and source records attributed to D Frenkel.

At least 37 records · Page 2Linked to original sources

PubMed2000

Modulation of Alzheimer's beta-amyloid neurotoxicity by site-directed single-chain antibody.

A single-chain antibody was constructed from variable regions of heavy and light genes of the parental anti-beta-amyloid peptide IgM 508 antibody. This antibody exhibits anti-aggregating properties, leading to disaggregation of Alzheimer beta-amyloid (betaA) fibrils and prevents its toxic effect on cultured PC-12 cells. Sequencing of the small antibody, namely 508 (Fv), revealed that the V(L) domain contained a cysteine residue in the complementary determining region (CDR)3 (residue 96) which affects its solubility and stability. The cysteine codon was replaced using SOE PCR, and one of the mutants obtained, namely 508F(Fv) (containing phenylalanine instead of cysteine), showed an increased storage stability and higher affinity compared to the wild type. Antibody 508F(Fv) prevents the neurotoxicity of betaA (90% cell viability) and disrupts the fibril structure of beta-amyloid (62% decrease in ThT fluorescence). The ability of antibody 508F(Fv) to dissolve already-formed betaA fibrils makes it a good candidate for intracellular expression and modulation of APP processing as the first step towards the production of therapeutic protection molecules for Alzheimer's disease treatment.

Alzheimer Disease↗

PubMed2000

Influence of vacancies on the melting transition of hard disks in two dimensions

We present the results of molecular dynamics simulations of two-dimensional (2D) hard disk systems in the vicinity of melting. The simulations are used to calculate the elastic constants, which can be used to estimate the location of the Kosterlitz-Thouless dislocation unbinding transition. Simulations on defect-free lattices indicate that this transition is expected to occur at essentially the same density as a first-order solid-isotropic transition and so it is not possible to rule out either a one step weak first-order transition between the solid and the isotropic fluid or a two step transition via a hexatic phase. Simulations performed on systems with vacancies indicate that the elastic constants are essentially unchanged at constant density. This result implies that vacancies have little influence on the melting of 2D hard disk solids.

Journal Article↗

PubMed2000

Enhanced stability of layered phases in parallel hard spherocylinders due to addition of hard spheres

There is increasing evidence that entropy can induce microphase separation in binary fluid mixtures interacting through hard particle potentials. One such phase consists of alternating two-dimensional liquidlike layers of rods and spheres. We study the transition from a uniform miscible state to this ordered state using computer simulations, and compare results to experiments and theory. We conclude the following: (1) There is stable entropy driven microphase separation in mixtures of parallel rods and spheres. (2) Adding spheres smaller than the rod length decreases the total volume fraction needed for the formation of a layered phase, and therefore small spheres effectively stabilize the layered phase; the opposite is true for large spheres. (3) The degree of this stabilization increases with increasing rod length.

Journal Article↗

PubMed2000

Phase behavior of model mixtures of colloidal disks and polymers

The phase behavior of model colloidal systems containing platelets and polymer is investigated using computer simulation and perturbation theory. For polymer coils with a diameter sigma(P) larger than 0.3sigma(D), where sigma(D) is the diameter of the platelets, isotropic fluid-fluid coexistence is observed, in addition to the usual isotropic-nematic transition for hard disk systems. For very small polymer coils (sigma(P)<0.1sigma(D)), a nematic-nematic demixing transition is observed, although this occurs at extremely high platelet density. The case of colloidal platelets and thin stiff rods is also examined. Demixing is observed in the isotropic phase for long rods and in the nematic phase for short rods, as for large and small polymer coils, respectively. However, the nematic-nematic demixing transition for platelet-rod mixtures occurs at a much lower platelet density than in the platelet-disk mixtures.

Journal Article↗

PubMed2000

Vaccination for the prevention and treatment of Alzheimer's disease.

In vitro studies showed that beta-amyloid peptide neurotoxicity correlates with the formation of fibrillar beta-amyloid and the variation in neurotoxic potency is related to the extent of peptide aggregation. Many efforts are being focused on the development of potent and selective inhibitors of amyloid formation in order to reduce the extent of their deposition and related neurotoxic effects. In our laboratory we are pioneering the idea that site-directed monoclonal antibodies (MAbs) can solubilize synthetic beta-amyloid aggregates. Production and performance of such antibodies by repeated injections of toxic human beta-amyloid fibrils into transgenic mice suggests the feasibility of vaccination against Alzheimer's disease (AD). Here we report the development of a novel immunization procedure for the production of effective antiaggregating beta-amyloid antibodies. The antigen is built from filamentous phages displaying the only four amino acids EFRH located at positions 3-6 of the beta-amyloid peptide found to be the main regulatory site of amyloid formation. Autoimmune antibodies are obtained by EFRH phage administration in guinea pigs, which exhibit human identity in the beta-amyloid peptide region. Availability of such antibodies opens up possibilities for the development of an efficient and long-lasting immunization procedure for the treatment of AD.

Journal Article↗

PubMed1999

High affinity binding of monoclonal antibodies to the sequential epitope EFRH of beta-amyloid peptide is essential for modulation of fibrillar aggregation.

Monoclonal antibodies raised against the N-terminal of Alzheimer's beta-amyloid peptide (betaAP) were found to modulate its fibrillar aggregation. While mAbs 6C6 and 10D5 inhibit the formation of beta-amyloid fibrils, trigger disaggregation and reversal to its non-toxic form, mAb 2H3 is devoid of these properties. MAb 2H3 binds the sequence DAEFRHD, corresponding to position 1-7 of the betaAP with high affinity (2 x 10(-9) M) similar to its binding with the whole betaAP. The EFRH peptide strongly inhibits binding of mAbs 6C6 and 10D5 to betaAP, whereas it inhibits weakly the interaction of 2H3 with betaAP. Low affinity binding of mAb 2H3 to EFRH might explain its failure in prevention of beta-amyloid formation.

Amino Acid Sequence↗

PubMed1999

Matrix-assisted refolding of single-chain Fv- cellulose binding domain fusion proteins.

We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form. The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli. The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety. The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use. This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured. Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix. The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs. The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression.

Amino Acid Sequence↗

PubMed1999

Hypothyroidism protects rat liver from acetaminophen hepatotoxicity.

Recent data from animal studies suggest that induced hypothyroidism inhibits the development of liver injury in several animal models, including liver cirrhosis and fulminant hepatic failure in rats, and immune-mediated acute liver injury in mice. The aim of the present study was to determine whether hypothyroidism would likewise prevent acetaminophen-induced hepatic damage in rats. Liver damage was induced by acetaminophen (2 g/kg) administered by gavage to fasting rats as a single dose. Hypothyroidism was induced by methimazole, propylthiouracil, or surgical thyroidectomy and confirmed by elevated serum levels of TSH. Hypothyroidism significantly inhibited acetaminophen-induced liver damage as manifested by the decreased serum levels of liver enzymes, malondialdehyde and blood ammonia, as well as by the higher hepatic glutathione content, in all three groups of hypothyroid rats compared to euthyroid controls (P < 0.01). Histopathologic analysis showed significantly less liver necrosis and inflammation in the acetaminophen-treated hypothyroid rats. Oxygen extraction, measured in isolated perfused rat liver preparation, was also reduced in the hypothyroid livers to 42+/-8% compared to 81+/-14% of controls (P < 0.01). However, the expression of CYP2E1 in the livers of hypothyroid rats, as measured by western blot analysis, was not decreased compared to control rats. These results suggest that induced hypothyroidism, regardless of the mode of induction, protects rat liver from acetaminophen hepatotoxicity. This effect may be related to hypometabolism of liver cells, but the exact mechanism needs further clarification.

Acetaminophen↗

PubMed1998

N-terminal EFRH sequence of Alzheimer's beta-amyloid peptide represents the epitope of its anti-aggregating antibodies.

Monoclonal antibodies 6C6 and 10D5 raised against the N-terminal of beta-amyloid peptide interfere with the formation of beta-amyloid and trigger reversal to its non-toxic components. The epitopes of these antibodies were localized employing a library composed of filamentous phage displaying random combinatorial hexapeptides. Among 44 positive phage-clones, selected from the library by both antibodies, 40 clones carried the consensus sequence EFRH. These EFRH phage-clones bind specifically mAbs 6C6 or 10D5 with an apparent binding constant of approximately 10(-9) M. The peptide EFRH inhibits binding of mAbs 6C6 or 10D5 to beta-amyloid peptide in affinities identical to those obtained. with the peptides corresponding to positions 1-9, 1-16 and 1-40 of beta-peptide. These findings confirm that the peptide EFRH which is located at positions 3-6 within beta-amyloid peptide represents the sequential epitope of mAbs 6C6 and 10D5.

Amino Acid Sequence↗

PubMed1998

Pentoxifylline prevents concanavalin A-induced hepatitis by reducing tumor necrosis factor alpha levels and inhibiting adhesion of T lymphocytes to extracellular matrix.

BACKGROUND/AIMS: Concanavalin A activates T lymphocytes and causes T cell-mediated hepatic injury in mice. Tumor necrosis factor alpha is a critical mediator in this experimental model. T-cell-mediated liver injury involves the migration of immune cells, notably CD4+ T lymphocytes, into liver tissue. Pentoxifylline is a strong suppressor of tumor necrosis factor alpha release and prevents leukocyte adherence to vascular endothelium and down-regulates the expression of intercellular adhesion molecule-1 in monocytes. In this study, we examined the efficacy of pentoxifylline as a potential therapeutic compound for the treatment of concanavalin A hepatitis. METHODS: Balb/c mice were injected with 12 mg/kg concanavalin A with or without a single injection of pentoxifylline (5-300 mg/kg), 2 h prior to concanavalin A administration. Liver damage was evaluated by determining serum levels of liver enzymes and tumor necrosis factor alpha, and hepatic histopathology compared to mice treated with concanavalin A only. We also assessed the effects of pentoxifylline on the adhesive properties of T lymphocytes to fibronectin, as a paradigm for immune cell-extracellular matrix interactions required for migration. Pretreatment with pentoxifylline significantly reduced serum levels of liver enzymes (3800+/-650 vs 150+/-28 IU/l) and tumor necrosis factor alpha (710+/-105 vs 113+/-15 pg/ml) with no evidence of inflammation in histopathologic examination compared to control mice treated with concanavalin A. Pentoxifylline also inhibited the binding of murine T cells to fibronectin. All the effects of pentoxifylline were dose-dependent. CONCLUSIONS: These results indicate that high doses of pentoxifylline can prevent concanavalin A hepatitis by suppression of tumor necrosis factor alpha release and inhibition of T cells adhesion to extracellular matrix.

Animals↗

PubMed1997

Enhancement of protein crystal nucleation by critical density fluctuations.

Numerical simulations of homogeneous crystal nucleation with a model for globular proteins with short-range attractive interactions showed that the presence of a metastable fluid-fluid critical point drastically changes the pathway for the formation of a crystal nucleus. Close to this critical point, the free-energy barrier for crystal nucleation is strongly reduced and hence, the crystal nucleation rate increases by many orders of magnitude. Because the location of the metastable critical point can be controlled by changing the composition of the solvent, the present work suggests a systematic approach to promote protein crystallization.

Colloids↗

PubMed1996