Rafael Apitz-Castro

Ajoene, el principal compuesto activo derivado del ajo (Allium sativum), un nuevo agente antifúngico

Resumen Desde tiempos inmemoriales el ajo ha transitado junto al hombre por los caminos de la tierra, multiples son los beneficios que la humanidad ha obtenido de sus propiedades medicinales y magico-religiosas, pero fue en las ultimas tres decadas cuando el estudio de sus propiedades permitio establecer una base racional para sus potencialidades clinicas. Se conocio la existencia de la alicina, el ajoene, los tiosulfinatos y una gama de compuestos organosulfurados como componentes esenciales de este bulbo y se comprobo, para muchos de ellos, una diversidad de efectos que abarcan desde propiedades antitromboticas, antitumorales, antiparasitarias y antifungicas. Sin embargo, el ajoene, un producto de gran estabilidad, que se origina de la ruptura y la reparacion no enzimatica de la alicina, y que puede tambien ser obtenido sinteticamente, demostro poseer la mayor actividad biologica en todos los sistemas estudiados. En este articulo nos referiremos a las propiedades antifungicas del ajoene y a las potencialidades que posee esta novel molecula para ser utilizada en la terapeutica de las infecciones micoticas.

The storage pool deficiency in platelets from humans with the Chédiak-Higashi syndrome: study of six patients

Summary Functional and biochemical studies of platelets from human Chédiak‐Higashi syndrome (CHS) are scarce and/or incomplete. In the present report, the aggregation response to a variety of inducers of platelet aggregation, the content of the dense granule constituents ATP, ADP, serotonin and calcium, the secretion of ATP, ADP, and calcium induced by thrombin, the total content of magnesium, the incorporation of 14C‐adenine in the cytoplasmic pool of adenine nucleotides, as well as the content of intracellular cyclic‐AMP, have been quantitated in six patients with CHS. Furthermore, data is presented on the kinetics of uptake of radiolabelled serotonin and its storage in human CHS platelets. An abnormal aggregation behaviour was found in all patients. However, the response of CHS platelets to the different inducers studied did not show a uniform pattern. The total content and the maximal amounts of the dense granule constituents secretable by thrombin were greatly decreased in all six patients. Total magnesium content was similar to that of normal platelets. The ATP/ADP ratio was higher than in controls. Uptake of radiolabelled serotonin by CHS platelets closely followed the uptake by normal platelets; during the first 2–3 min, however, incorporation of the amine by CHS platelets came rapidly to a plateau which contrasts with the steady, linear increase in uptake found in controls. CHS platelets loaded with radiolabelled serotonin and gel‐filtered, showed a spontaneous release of radioactivity not observed in normal platelets under the same conditions. The cyclic‐AMP content of CHS platelets was similar to that of normals. In contrast to platelets from patients with storage pool disease, the secretable calcium from CHS platelets represents a 67% of total platelet

Ajoene, a garlic compound, inhibits protein prenylation and arterial smooth muscle cell proliferation.

Ajoene is a garlic compound with anti‐platelet properties and, in addition, was shown to inhibit cholesterol biosynthesis by affecting 3‐hydroxy‐3‐methyl‐glutaryl coenzyme A (HMG‐CoA) reductase and late enzymatic steps of the mevalonate (MVA) pathway. MVA constitutes the precursor not only of cholesterol, but also of a number of non‐sterol isoprenoids, such as farnesyl and geranylgeranyl groups. Covalent attachment of these MVA‐derived isoprenoid groups (prenylation) is a required function of several proteins that regulate cell proliferation. We investigated the effect of ajoene on rat aortic smooth muscle cell proliferation as related to protein prenylation. Cell counting, DNA synthesis, and cell cycle analysis showed that ajoene (1–50 μM) interfered with the progression of the G1 phase of the cell cycle, and inhibited rat SMC proliferation. Similar to the HMG‐CoA reductase inhibitor simvastatin, ajoene inhibited cholesterol biosynthesis. However, in contrast to simvastatin, the antiproliferative effect of ajoene was not prevented by the addition of MVA, farnesol (FOH), and geranylgeraniol (GGOH). Labelling of smooth muscle cell cellular proteins with [3H]‐FOH and [3H]‐GGOH was significantly inhibited by ajoene. In vitro assays for protein farnesyltransferase (PFTase) and protein geranylgeranyltransferase type I (PGGTase‐I) confirmed that ajoene inhibits protein prenylation. High performance liquid chromatography (HPLC) and mass spectrometry analyses also demonstrated that ajoene causes a covalent modification of the cysteine SH group of a peptide substrate for protein PGGTase‐I. Altogether, our results provide evidence that ajoene interferes with the protein prenylation reaction, an effect that may contribute to its inhibition of SMC proliferation.

Expression of biological activity of draculin, the anticoagulant factor from vampire bat saliva, is strictly dependent on the appropriate glycosylation of the native molecule

Draculin, a glycoprotein isolated from vampire bat (Desmodus rotundus) saliva, is a natural anticoagulant which inhibits activated coagulation factors IX (IXa) and X (Xa). The observation that under captivity conditions, the anticoagulant activity present in vampire bat saliva is dependent upon the salivation protocol, led us to investigate the possible relationship between the expression of biological activity of native draculin and the post-translational glycosylation of the protein backbone. Daily salivation of vampire bats yields a saliva that progressively decreases in anticoagulant activity, without any significant change in overall protein content, or in the amount of protein specifically recognized by a polyclonal anti-draculin antibody. Anticoagulant activity of the saliva is restored after a 4-day period of rest. Besides the marked difference in anticoagulant activity, purified native draculin, obtained from high- and low-activity saliva, shows significant differences in: (a) composition of the carbohydrate moiety, and (b) Glycosylation pattern. Furthermore, controlled chemical deglycosylation of native draculin, under conditions that do not affect the polypeptide backbone, progressively leads to complete loss of the biological activity. Our present results implicate that correct glycosylation of draculin is a seminal event for the expression of the biological activity of this glycoprotein.

The phospholipase A2 from human platelets

Studies on a purified phospholipase A2 (PLA2) from human platelets show that the enzyme, which is copurified with the plasma membrane fraction, has a MW of ∼50 K Dalton, requires Ca++, and has a pH optimum of 9.4. Under optimal conditons, PLA2 activity corresponds to at least 13 nmol/min/109 platelets. Unsaturated PL are preferred substrates and the enzyme is considerably more active on the aggregated form of the substrate than on the monomers. The specific activity is markedly affected by the quality of the interface, showing variations of more than 10-fold between different substrate forms. In the absence of detergents, a 4-fold increase in rate is observed when both products are present. Maximal rates are obtained at 20 mole percent of products to substrate. 1,2-Diglyceride and phosphatidic acid stimulate the hydrolysis of PC by the purified enzyme, however, in these forms of the substrate, neither of them are hydrolyzed. Activation of this enzyme by some intermediate of the phospholipase C pathway might play a role in the stimulus-linked release of platelet arachidonic acid.

Atherogenicity of Hypercholesterolemia in the Precense of Hemolysis in Spite of Heme Oxygenase-1 Induction

Atherosclerosis appears to be caused mainly by a focal and complex tissue response to apoB-containing lipoproteins (LDL, IDL, VLDL) deposition in the arterial intima (Camejo et al., 1998b; Williams and Tabas, 1995). The retention of apoBlipoproteins by proteoglycans of the arterial intima seems to provide the opportunity for enzymatic and non-enzymatic alterations of their lipid and protein moieties. This appears to cause formation of substances, mainly bioactive lipids that acting locally on arterial cells could initiate an inflammatory atherogenic response (Camejo et al., 1998b; Hurt-Camejo et al., 2001). Among the most studied potentially atherogenic modifications of lipoproteins are those caused by free radical-mediated reactions, probably taking place in the intima (Steinberg and Witztum, 1999). However, the origin of reactive oxygen species (ROS) that affect lipoproteins and cells in arteries still remains uncertain. The major cause for this is that agents, like transition metals, used as a model of pro-oxidant conditions in vitro do not oxidize lipoproteins in the presence of other plasma components that are known to exist in circulation and in the intima (Steinberg and Witztum, 1999). Heme is an iron-containing catabolic product of hemoglobin that has high affinity for human low density lipoprotein (LDL) in plasma and that is capable of oxidizing this particle in diluted plasma (Camejo et ah, 1998a; Tribble et al., 1995) Heme interacts with the lipid surface monolayer and with specific positively charged segments of apoB-100 in the LDL particle (Camejo et al, 1998a). Heme transported by apoB-lipoproteins can accumulate in the arterial intima and become an in situ generator of ROS.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A2

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.