Amyloid Precursor Protein and Tau Peptides Linked Together Ameliorate Loss of Cognition in an Alzheimer's Disease Animal Model.

The major proteins involved in Alzheimer's disease (AD) are amyloid precursor protein (APP) and Tau. We demonstrate that APP1 (390-412) and Tau1 (19-34), linked together with either a flexible or a rigid peptide bridge, are able to inhibit, in vitro, the interaction between APP and Tau proteins. Furthermore, nasal administration of biotin-labelled Flex peptide for two weeks indicated the localization of the peptide around and close to plaques in the hippocampus area. In vivo studies in 5xFAD transgenic (Tg) mice, which exhibit plaque load and mild cognitive decline at four months of age, show that nasal administration of the flexible linked peptide reduced amyloid plaque burden. Additionally, nasal treatment with either flexible or rigid linked peptides prevented cognitive function deterioration. A significant treatment effect was achieved when either treatment was initiated at the age of three months, before severe cognitive deficiency is evident, or at five months, when such deficiency is already observed. The nasally treated mice demonstrated a cognitive ability not significantly different from the non-Tg littermate controls. Testing the effect of the flexible peptide by gavage feeding on the cognitive function of 5xFAD Tg mice demonstrated that feeding as well as nasal treatment significantly improves the cognitive ability of Tg mice compared to control PBS-treated mice.

Peptide Interference with APP and Tau Association: Relevance to Alzheimer's Disease Amelioration.

The two major proteins involved in Alzheimer's disease (AD) are the amyloid precursor protein (APP) and Tau. Here, we demonstrate that these two proteins can bind to each other. Four possible peptides APP1 (390-412), APP2 (713-730), Tau1 (19-34) and Tau2 (331-348), were predicted to be involved in this interaction, with actual binding confirmed for APP1 and Tau1. In vivo studies were performed in an Alzheimer Disease animal model-APP double transgenic (Tg) 5xFAD-as well as in 5xFAD crossed with Tau transgenic 5xFADXTau (FT), which exhibit declined cognitive reduction at four months of age. Nasal administration of APP1 and Tau1 mixture, three times a week for four or five months, reduced amyloid plaque burden as well as the level of soluble Aß 1-42 in the brain. The treatment prevented the deterioration of cognitive functions when initiated at the age of three months, before cognitive deficiency was evident, and also at the age of six months, when such deficiencies are already observed, leading to a full regain of cognitive function.

A sensitive colorimetric determination of cyanuric chloride and its activated agarose immobilization resins.

A colorimetric method for determining cyanuric chloride (CC) and for monitoring its polysaccharide gel activation, before and after ligand binding, was developed. The method is based on the reaction of CC or its activated gel with pyridine and barbituric acid or dimethylbarbituric acid. The product formed yields a purple red color with λ max at 595 nm, and an EM value of approximately 300,000 after 1 h at room temperature. Due to its high sensitivity, this method can detect traces of CC (1 µM) under the above conditions. The method is fast, reliable and devoid of any side reactions.

Mussel-inspired new approach for polymerization and cross-linking of peptides and proteins containing tyrosines by Fremy's salt oxidation.

Our objective was to develop a method mimicking the natural process of coherence in marine mollusks, by direct chemical conversion of protein tyrosine residues to DOPA-o-quinones, which consequently generates polymerization and cross-linking. Fremy's salt, (ON(SO3K)2, was used to convert tyrosine residues in peptides and proteins to reactive o-quinones. The conversion of tyrosines to DOPA-o-quinones, and their ability to polymerize or cross-link, was tested on tyramine, peptides, and proteins. The peptides tested were as follows: biotin-PEG4-tyramine (PEG-BT), and two decapeptides (identical to the repeating units comprising the mussel's adhesive protein). The proteins tested were as follows: bovine pancreatic ribonuclease A (RNase), lysozyme, IgG, avidin, and streptavidin. The oxidized peptides and proteins were all shown to incorporate oxygen atoms and undergo polymerization and cross-linking, depending on the availability of nucleophiles, mostly lysine amino groups of proteins. All the peptides and the noninteracting proteins such as RNase and lysozyme underwent homopolymerization upon Fremy's salt oxidation. When Fremy's salt oxidaized PEG-BT was mixed with the above proteins, it did not react with any of these proteins because PEG-BT underwent fast self-polymerization. Conversely, streptavidin or avidin cross-linked with PEG-BT after preincubation, thus showing that biorecognition is a prerequisite for cross-linking. Polymerization and cross-linking also occurred, following Fremy's salt oxidation of interacting proteins such as avidin and strepavidin with biotinyilated lysozyme or biotinylated RNase. This indicates that only proteins in very close proximity readily cross-link and polymerize via tyrosine residues. Attempts to convert DOPA-quinone to DOPA by reduction with sodium dithionite (Na2S2O4), was successful as far as small peptides were used. Fremy's salt oxidation can serve as an easy and useful tool to polymerize and cross-link proteins, for fabrication of biomaterials and to study protein-protein interactions.

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein.

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal ß-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.

Allicin up-regulates cellular glutathione level in vascular endothelial cells.

BACKGROUND: Allicin in garlic is the primary active compound known to rapidly interact with free thiols. AIMS OF THE STUDY: To examine the effect of allicin on gene expression and glutathione cellular level in vascular endothelial cells. METHODS: Cultured endothelial cells were exposed to allicin; mRNA was prepared and subjected to Micro-array and Real-Time PCR. Glutathione cellular level was determined on cell lysates. RESULTS: Micro-array analysis demonstrated allicin-induced up- and down-regulation of 116 and 100 genes, respectively. Up-regulated genes included the phase II detoxifying enzymes thioredoxin reductase 1 and 2, heme oxygenase-1 and glutamate cysteine lygaze modifier subunit, the rate limiting enzyme in glutathione biosynthesis. Endothelial cells exposed to allicin and its derivatives containing glutathione or cysteine residues increased cellular glutathione. Allicin increased the glutathione level in a concentration and time-dependent manner up to 8-fold at a concentration of 10-20 microM after 28 h exposure. Furthermore, allicin derivative-treated cultures demonstrated a 50% decrease in tBuOOH cytotoxicity. CONCLUSIONS: These results may suggest a putative role for allicin and its derivatives in preventing reactive oxygen species damage by up-regulating the phase II detoxifying enzymes and increasing the cellular glutathione level.

Two structures of alliinase from Alliium sativum L.: apo form and ternary complex with aminoacrylate reaction intermediate covalently bound to the PLP cofactor.

Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent alpha, beta-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the epsilon-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells.

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.

Epitope identification of specific naturally occurring human anti-avidin antibodies.

Human serum contains natural antibodies against avidin. Affinity purified natural anti-avidin human IgG exhibits affinity constants comparable to those of antibodies produced by active immunization of rabbits. Using a random hexapeptide library displayed on the filamentous M13 phage, and rabbit anti-avidin purified antibodies as a selector, we searched for epitopes shared by both selector and natural human anti-avidin IgG. This approach, enabled the isolation and identification of phagotopes bearing consensus motifs similar to sequence stretches of the avidin loops and ß-sheet regions. These phagotopes were recognized by the natural human anti-avidin antibodies. The fact that natural anti-avidin antibodies in human serum have similar epitopes to those of IgG elicited by active immunization of animals, led us to suggest that small peptide epitopes may prevent deleterious effects caused by antibodies formed against food proteins as well as therapeutic proteins.

Factors dictating the pseudocatalytic efficiency of avidins.

The hydrolysis of biotinyl p-nitrophenyl ester (BNP) by a series of avidin derivatives was examined. Surprisingly, a hyperthermostable avidin-related protein (AVR4) was shown to display extraordinary yet puzzling hydrolytic activity. In order to evaluate the molecular determinants that contribute to the reaction, the crystal structure of AVR4 was compared with those of avidin, streptavidin and key mutants of the two proteins in complex with biotinyl p-nitroanilide (BNA), the inert amide analogue of BNP. The structures revealed that a critical lysine residue contributes to the hydrolysis of BNP by avidin but has only a minor contribution to the AVR4-mediated reaction. Indeed, the respective rates of hydrolysis among the different avidins reflect several molecular parameters, including binding-site architecture, the availability of the ligand to solvent and the conformation of the ligand and consequent susceptibility to efficient nucleophilic attack. In avidin, the interaction of BNP with Lys111 and disorder of the L3,4 loop (and consequent solvent availability) together comprise the major driving force behind the hydrolysis, whereas in AVR4 the status of the ligand (the pseudo-substrate) is a major distinguishing feature. In the latter protein, a unique conformation of the L3,4 loop restrains the pseudo-substrate, thereby exposing the carbonyl carbon atom to nucleophilic attack. In addition, due to its conformation, the pseudo-substrate in the AVR4 complex cannot interact with the conserved lysine analogue (Lys109); instead, this function is superseded by polar interactions with Arg112. The results demonstrate that, in highly similar proteins, different residues can perform the same function and that subtle differences in the active-site architecture of such proteins can result in alternative modes of reaction.

Essentials of biorecognition: the (strept)avidin-biotin system as a model for protein-protein and protein-ligand interaction.

Molecular recognition or biorecognition is as the heart of all biological interactions. These interactions are characterized by a collection of noncovalent bonds, namely ionic, hydrogen-bonding and hydrophobic interactions. In addition, shape complementarity appears to play a pivotal role in the process of biorecognition. In this review, we examine the versatile avidin-biotin complex as a model system for study of the biorecognition phenomenon with respect to protein-protein, protein-peptide, protein-ligand and protein-DNA interactions.

Apoptotic killing of B-chronic lymphocytic leukemia tumor cells by allicin generated in situ using a rituximab-alliinase conjugate.

Allicin, a highly active component from freshly crushed garlic, is produced upon the reaction of the small molecular weight molecule alliin, with the enzyme alliinase (EC 4.4.1.4). Because allicin was shown to be toxic to various mammalian cells in vitro, we devised a novel approach for the therapy of B-cell malignancies based on site-directed generation of allicin. Alliinase was conjugated to the monoclonal antibody rituximab, which recognizes the CD20 antigen, and the resulting conjugate was targeted to CD20+ B chronic lymphocytic leukemia (B-CLL) and other B-cell lymphomas. Upon addition of alliin, allicin was formed in situ, killing the CD20+ tumor B cells via apoptosis. Following a 72-hour treatment, an 85% and 96% reduction was observed in the number of viable B-CLL and EBV-transformed B cells, respectively. Using the human/mouse radiation chimera for the evaluation of allicin targeting in a preclinical animal model, we showed a significant reduction in the number of recovered B-CLL, mantle cell lymphoma, or EBV-transformed B cells. We conclude that our system offers a new powerful and less toxic therapy for B-CLL and other B-cell malignancies. Furthermore, combining alliinase with the appropriate monoclonal antibody may extend the application of this approach to other conditions in which the elimination of a specific cell population is desired.

The effects of S-allylmercaptocaptopril, the synthetic product of allicin and captopril, on cardiovascular risk factors associated with the metabolic syndrome.

Pure allicin, prepared biosynthetically by reacting synthetic alliin with an immobilized alliinase enzyme, is known to possess cardioprotective effects. However, in its pure form, allicin is pharmacologically unstable. S-allylmercaptocaptopril (CPSSA) is a new stable synthetic compound produced by chemical reaction between allicin and the angiotensin converting enzyme inhibitor captopril. Using the fructose-induced metabolic syndrome rat model we studied the effects of short-term treatment with two doses of CPSSA on cardiovascular risk factors associated with the metabolic syndrome, in comparison to the effects of allicin and captopril separately. Allicin (8 mg/(kg day)) significantly reduced insulin, triglycerides, and homocysteine concentrations, and had a slight effect on SBP. Captopril (50mg/(kg day)) only improved blood pressure and homocysteine. Treatment with low dose of CPSSA (5mg/(kg day)) lowered SBP but did not improve any other measured parameter, while treatment with a higher dose (50mg/(kg day)) significantly decreased blood pressure, triglycerides, and homocysteine concentrations. We conclude that the combined molecule CPSSA integrates the anti-hypertensive, lipid-lowering, and homocysteine-reducing effects of both allicin and captopril, making it a potential cardiovascular protective agent.

Inhibition of tumor growth by a novel approach: in situ allicin generation using targeted alliinase delivery.

Allicin (diallyl thiosulfinate), a highly active component in extracts of freshly crushed garlic, is the interaction product of non-protein amino acid alliin (S-allyl-L-cysteine sulfoxide) with the enzyme alliinase (alliin lyase; EC 4.4.1.4). Allicin was shown to be toxic in various mammalian cells in a dose-dependent manner in vitro. We made use of this cytotoxicity to develop a novel approach to cancer treatment, based on site-directed generation of allicin. Alliinase from garlic was chemically conjugated to a mAb directed against a specific tumor marker, ErbB2. After the mAb-alliinase conjugate was bound to target tumor cells, the substrate, alliin, was added. In the presence of alliin, tumor-localized alliinase produced allicin, which effectively killed N87 and CB2, both ErbB2-expressing cells in vitro, whereas 32D cells (a murine hematopoietic progenitor cell line, devoid of the ErbB2 receptors) were not affected. Moreover, using N87, a human tumor cell line xenograft in athymic nude mice, we demonstrated for the first time, a high antitumor activity of allicin that was produced in situ by the conjugate, on alliin administration in vivo, while at the same time other tissues were unharmed due to the inert nature of alliin and the high clearance rate of allicin. The effect of the treatment on tumor growth arrest became significant 2 weeks after its onset, and it continued to rise, reaching highly significant inhibition a week later. Ten days after the end of the treatment (day 18), tumor growth inhibition was still the same.

Allylmercaptocaptopril: a new antihypertensive drug.

Allylmercaptocaptopril (CPSSA) was synthesized by reacting captopril with pure allicin. Fructose-induced hypertensive groups of rats were fed a fructose-rich diet for 3 weeks, and then received the diet plus either CPSSA (40 to 56 mg or 138 to 194 micromol/L/kg/d) or captopril (80 mg or 369 micromol/L/kg/d) for 2 more weeks. CPSSA (both doses) significantly lowered blood pressure (BP) from 153.4 to 120.8 mm Hg (P <.005). Captopril gave similar results, lowering BP from 150.7 to 123 mm Hg (P <.005). CPSSA also decreased the high levels of triglycerides to normal. The new stable compound allylmercaptocaptopril combines the beneficial properties of captopril and allicin and is a potential candidate for antihypertensive drug therapy.

The effects of allicin on weight in fructose-induced hyperinsulinemic, hyperlipidemic, hypertensive rats.

BACKGROUND: Commercially available garlic preparations in the form of garlic oil, garlic powder and pills are widely used for certain therapeutic purposes, including lowering blood pressure and improving lipid profile. Despite the impressive effects of garlic most studies are limited by lack of controlled methods and suitable double-blinding, and by the use of preparations with unknown amounts and chemical identification of the active ingredient. Allicin, a synthetic preparation of an active constituent of garlic, was found to lower blood pressure, insulin, and triglycerides levels in fructose-fed rats. Thus, it was considered important to assess its effect on the weight of the animals. METHODS: Male Sprague-Dawley rats weighing 240 to 250 g were fed a fructose-enriched diet consisting of 21% protein, 5% fat, 60% carbohydrate, 0.49% sodium and 0.49% potassium for 5 weeks, which produced hyperinsulinemia, hypertension, and hypertriglyceridemia. Group I (controls) rats were fed a diet enriched by fructose only; group II was given allicin the last 2 weeks, and group III was given allicin the first 3 weeks. The three groups consumed the same amount of food. Weight was measured at the beginning of the experiment and after 3 and 5 weeks on the diet. RESULTS: Weight in the control group rose from 249.4 +/- 10.04 g to 274.5 +/- 15.5 g after 3 weeks and to 306.9 +/- 22.2 g after 5 weeks. Weight in group II rose from baseline 259.1 +/- 12.1 g to 306.9 +/- 22.2 g after 3 weeks on fructose alone, and declined slightly to 282.4 +/- 17.4 g after 2 weeks of allicin (P <.02). In group III, in which the protocol was reversed, the baseline weight of 260.4 +/- 13.25 g rose only to 269.8 +/-15.3 g (P <.431) after 3 weeks on fructose and allicin. CONCLUSIONS: The control group that was fed a diet enriched by fructose alone continued to gain weight, whereas the groups fed allicin did not. The difficulty of preventing weight gain after reaching the nadir of weight loss underscores the practical value of allicin for weight control.

Oriented versus random protein immobilization.

Immobilization of proteins on solid surfaces plays an important role in all the fields of modern biology. Two approaches are used in the immobilization of proteins: a random and an oriented mode of binding to solid matrices. In this note, we show that there is not much difference in using either mode of immobilization, since proteins usually bind to a matrix through only one or two bonds. This is demonstrated by the attachment of several proteins to CNBr-activated Sepharose through their lysines and the consequent conversion of those lysines to homoarginine upon treatment with ammonium hydroxide.

S-allyl derivatives of 6-mercaptopurine are highly potent drugs against human B-CLL through synergism between 6-mercaptopurine and allicin.

S-allylthio-6-mercaptopurine and its ribose derivative were tested for anti-leukemic activity, using a human- mouse B-CLL model. The novel prodrugs contain two components, a purine analog, which interferes with DNA synthesis, and an S-allylthio, readily engaging in thiol-disulfide exchange reactions. The latter component targets the redox homeostasis which is more sensitive in leukemic cells, than in normal B-cells. Upon administration, the prodrug permeates cells, instantly reacts with free thiol, forming S-allyl mixed disulfides and releasing purine. Several cycles of thiol-disulfide exchange reactions occur, thus extending the duration of the prodrug effects. The concerted action of 2 components, as compared with purine alone, boosted in vitro apoptotis in B-CLL cells from 10% to 38%, and decreased in vivo engraftment of B-CLL from 30% to 0.7%.

Reaction mechanisms of allicin and allyl-mixed disulfides with proteins and small thiol molecules.

Allylsulfides from garlic are chemopreventive agents. Entering cells they are expected to initially interact with glutathione. Accordingly, reaction mechanisms of the product, S-allylthio-glutathione, with model proteins and thiols were analyzed in cell free systems. With glutathionyl, cysteinyl or captopril representing S-allyl aliphatic adducts, the reaction with sulfhydryl groups resulted in mixed disulfide mixtures, formed by both, S-allyl and aliphatic moieties. To improve conventional prodrug treatment of blood pressure, cancer and intestinal inflammation S-allylthio prodrugs, such as S-allylthio-6-mercaptopurine and S-allylthio-captopril were synthesized. Synergistic activities of the 2 constituents, as well as increased cell permeability allow for efficient in vivo activity. Upon reaction of these derivatives with glutathione, S-allylthio-glutathione is formed, while 6-mercaptopurine is the leaving group. Excess cellular glutathione enables several cycles of sulfhydryl-disulfide exchange reactions to occur, extending the hybrid drug's pharmacodynamics.