Kailash C. Agarwal

Therapeutic actions of garlic constituents

Most studies on garlic during the past 15 years have been primarily in the fields of cardiovascular and cancer research. Cardiovascular studies have been mainly related to atherosclerosis, where effects were examined on serum cholesterol, LDL, HDL, and triglycerides. Although the studies were not consistent in relation to the dosage, standardization of garlic preparations, and period of treatment, most findings suggest that garlic decreases cholesterol and triglycerides levels in patients with increased levels of these lipids. Lowering of serum lipids by garlic ingestion may decrease the atherosclerosis process. The other major beneficial effect of garlic is due to its antithrombotic actions. This field of garlic research has been extensively studied. Garlic extracts and several garlic constituents demonstrate significant antithrombotic actions both in vitro and in vivo systems. Allicin and adenosine are the most potent antiplatelet constituents of garlic because of their in vitro effects. Since both allicin and adenosine are rapidly metabolized in human blood and other tissues, it is doubtful that these compounds contribute to any antithrombotic actions in the body. In addition, ajoene also seems not to be an active antiplatelet principle, because it is not naturally present in garlic, garlic powders, or other commercial garlic preparations. Only a small amount of ajoene can be found in garlic oil-macerates; however, ajoene is being developed as a drug for treatment of thromboembolic disorders. Recent findings on the identification of potent enzyme inhibiting activities of adenosine deaminase and cyclic AMP phosphodiesterase in garlic extracts are interesting, and may have a significant role in the pharmacological actions in the body. Presence of such enzyme inhibitors in garlic may perhaps explain several clinical effects in the body, including the antithrombotic, vasodilatory, and anticancer actions. Epidemiological studies have suggested that garlic plays a significant role in the reduction of deaths caused by malignant diseases. This had led many investigators to examine garlic and garlic constituents for their antitumor and cytotoxic actions both in vitro and in laboratory animals. The data from these investigations suggest that garlic contains several potentially important agents that possess antitumor and anticarcinogenic properties. In summary, the epidemiological, clinical, and laboratory data have proved that garlic contains many biologically and pharmacologically important compounds, which are beneficial to human health from cardiovascular, neoplastic, and several other diseases. Numerous studies are in progress all over the world to develop effective and odorless garlic preparations, as well as to isolate the active principles that may be therapeutically useful.

Identification and isolation on a large scale of guanylate kinase from human erythrocytes: Effects of monophosphate nucleotides of purine analogs

Abstract Guanylate kinase has been identified and partially purified from human erythrocytes. A simple method has been developed, using calcium phosphate gel and DEAE-cellulose, for the purification of guanylate kinase and several other erythrocytic enzymes such as purine nucleoside phosphorylase, nucleoside diphosphokinase, adenylate kinase, lactic dehydrogenase, pyruvate kinase and phosphoglucose isomerase. The method is adaptable for both small and large scale purification and has been applied successfully to as much as 40 to 50 1. of human blood. Studies with the partially purified erythrocytic guanylate kinase showed that GMP, dGMP, 8-azaGMP and IMP serve as substrates while UMP, CMP and 6-thioIMP do not. 6-ThioGMP was not a substrate but was a competitive inhibitor with GMP with a K 1 of 7·5 × 10 −5 M. Preliminary findings suggest the occurrence of several isozymes of this enzyme. Other kinetic parameters of the enzyme are described.

INCORPORATION OF ANALOG PURINE NUCLEOSIDES INTO THE FORMED ELEMENTS OF HUMAN BLOOD: ERYTHROCYTES, PLATELETS, AND LYMPHOCYTES*

Analogs of purine and pyrimidine nucleosides have received considerable attention in many laboratories concerned with the development of chemotherapeutic agents for malignancies, viruses, and tropical parasites. In addition, a number of these compounds have shown important immunosuppressive activity or have proved of use for the treatment of diseases such as psoriasis. For the past few years, this laboratory has been interested in the interaction of natural or analog purine nucleosides with the formed elements of human blood, i.e., erythrocytes, platelets and lymphocytes. Although these studies were designed originally to analyze the pathways of purine nucleoside metabolism in these cells, a number of unexpected observations have been made that have not only broadened our understanding of normal cellular functions but that also suggest possible useful applications. This report summarizes a number of investigations that have been published earlier or that will be published in greater detail in the future.

Pathways of nucleotide metabolism in Schistosoma mansoni—III: Identification of enzymes in cell-free extracts☆

Abstract A survey of purine anabolic and catabolic enzymes has resulted in identification of major pathways in schistosome nucleotide biosynthesis. It is shown that multiple pathways for the incorporation of purine bases and nucleosides exist. The evidence suggests that adenosine phosphoribosyltransferase (APRT) activity is about ten times greater than adenosine kinase activity. Furthermore adenosine is converted to AMP principally via the pathway of adenosine deaminase, followed by conversion of inosine to hypoxanthine. In this sequence hypoxanthine phosphoribosyltransferase (HPRT) activity is rate limiting. On the basis of enzyme activities determined, one can suggest candidates of nucleotide analogs which might be useful chemotherapeutic agents.

Treatment of metastasis

A method for reducing the incidence of metastasis in tumor victims without treating the primary tumor per se resides in the administration of forskolin or its analogs which are potent inhibitors of platelet aggregation. Forskolin compounds are generally defined as labdane diterpenoids having the general formula: ##STR1## These compounds may be administered alone or in combination with other inhibitors of platelet aggregation.

Inhibition of hepatic metastasis from a human pancreatic adenocarcinoma (RWP-2) in the nude mouse by prostacyclin, forskolin, and ketoconazole

Metastasis is a multistep phenomenon in which platelets appear to play an important role. This study examined several compounds for their effects on experimental hepatic metastasis and on human pancreatic tumor cell‐platelet interactions. Prostacyclin (PGI2) and forskolin (stimulators of platelet adenylate cyclase) and ketoconazole (inhibitor of lipoxygenese and thromboxane synthetase) were used in order to investigate their effects on hepatic metastases from a human pancreatic tumor cell (RWP‐2) in the nude mouse. The tumor cells were injected intrasplenically and the animals were divided into control, prostacyclin (PGI2 200 μg), forskolin (150 μg), and ketoconazole (180 μg) groups. All three drugs were administered intraperitoneally 30 minutes before and 24 hours after the tumor cell injections. Statistically significant differences were observed between control and treated groups in tumor surface area (P < 0.001), percentage of liver surface area occupied by tumor (P < 0.001), and number of tumor colonies (P < 0.004 for prostacyclin, P < 0.005 for forskolin, and P < 0.001 for ketoconazole). These agents also strongly inhibited RWP‐2‐induced platelet aggregation in human platelet‐rich plasma.

Studies on the biochemical actions of 6-selenoguanine and 6-selenoguanosine

Abstract Survival studies were performed in mice bearing Sarcoma 180 ascites tumor treated with 6-thio and 6-seleno analogs of guanine and guanosine. The selenium-containing analogs were somewhat superior to the sulfur-containing compounds in antitumor activity and therapeutic index. The formation of 6-SeGMP from 6-seleno-guanine (6-SeG) was demonstrated in Sarcoma 180 ascites cells. Guanine, 6-thioguanine (6-TG) and 6-SeG show comparable substrate activity whereas 8-azaguanine is a much poorer substrate for hypoxanthine-guanine phosphoribosyl transferase from Sarcoma 180 cells. Both 6-TG and 6-SeG are good substrates for purine nucleoside phosphorylase from Sarcoma 180 cells. Chemically and enzymatically synthesized 6-SeGMP behaved as a competitive inhibitor ( K i 1 × 10 −4 M) of erythrocytic and Sarcoma 180 guanylate kinases. Weak substrate activity was demonstrated in the presence of large amounts of erythrocytic guanylate kinase.

Effect of serotonin on cyclic nucleotides of human platelets.

Serotonin produced a 6 to 10 fold increase of cyclic GMP over baseline levels of this nucleotide in platelets. Maximum stimulation was reached within 30 sec to 1 min after addition of serotonin and was dependent upon its concentration in the medium. Inhibition of serotonin uptake by methysergide, dihydroergotamine and chloroimipramine did not influence the serotonin-induced stimulation of cyclic GMP but glutaraldehyde and formaldehyde blocked it completely. Cyclic AMP levels in platelets were not affected by serotonin. The serotonin-induced stimulation of cyclic GMP is independent of the uptake of this biogenic amine by platelets and is not due to platelet aggregation.

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.

Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects

The biochemical, ultrastructural and functional aspects of digitonin-permeabilized platelets were investigated. Human platelets were permeabilized by exposure to the steroid glycoside digitonin. A 60 microM concentration of this permeabilizer produced a very substantial release of cytosolic enzymes from the platelets. Release from subcellular granules was relatively low and did not inhibit the response of platelets to a series of agonists. Although digitonin-permeabilized platelets required higher threshold concentrations of the usual stimulants, both primary and secondary aggregation as well as the release of nucleotides and enzymes from their respective granules remained intact. Transmission electron micrographs revealed discontinuities in the plasma membrane of digitonin-treated platelets, but scanning electron microscopy showed no difference between control and permeabilized platelets. No substantial loss of structural or membrane proteins could be detected by one- and two-dimensional gel electrophoresis. The pore size produced by digitonin treatment was sufficient to allow entry of 125I-labeled IgG into the platelet cytosolic space.