Biomedical subjects

G A Rosenthal

Publications and source records attributed to G A Rosenthal.

At least 19 recordsLinked to original sources

PubMed2001

L-Canavanine: a higher plant insecticidal allelochemical.

L-Canavanine, L-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease. The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins. The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical.

Animals↗

PubMed1999

Growth inhibition of A549 human lung adenocarcinoma cells by L-canavanine is associated with p21/WAF1 induction.

L-Canavanine (CAV) is a higher plant nonprotein amino acid and a potent L-arginine antimetabolite. CAV can inhibit the proliferation of tumor cells in vitro and in vivo, but little is known regarding the molecular mechanisms mediating these effects. We demonstrated that the treatment of human lung adenocarcinoma A549 cells with CAV caused growth inhibition; G1 phase arrest is accompanied by accumulation of an incompletely phosphorylated form of the retinoblastoma protein, whose phosphorylation is necessary for cell cycle progression from G1 to S phase. In addition, CAV induces the expression of p53 and subsequent expression of a cyclin-dependent kinase inhibitor, p21/WAF1. The p53-dependent induction of p21/WAF1 and the following dephosphorylation of the retinoblastoma protein by CAV could account for the observed CAV-mediated G1 phase arrest.

Adenocarcinoma↗

PubMed1997

The biochemical basis for L-canavanine tolerance by the tobacco budworm Heliothis virescens (Noctuidae).

The tobacco budworm, Heliothis virescens (Noctuidae), a destructive insect pest, is remarkably resistant to L-canavanine, L-2-amino-4-(guanidinooxy)butyric acid, an arginine antimetabolite that is a potent insecticide for nonadapted species. H. virescens employs a constitutive enzyme of the larval gut, known trivially as canavanine hydrolase (CH), to catalyze an irreversible hydrolysis of L-canavanine to L-homoserine and hydroxyguanidine. As such, it represents a new type of hydrolase, one acting on oxygen-nitrogen bonds (EC 3.13.1.1). This enzyme has been isolated from the excised gut of H. virescens and purified to homogeneity; it exhibits an apparent Km value for L-canavanine of 1.1 mM and a turnover number of 21.1 micromol x min(-1)x micromol(-1). This enzyme has a mass of 285 kDa and is composed of two subunits with a mass of 50 kDa or 47.5 kDa. CH has a high degree of specificity for L-canavanine as it cannot function effectively with either L-2-amino-5-(guanidinooxy)pentanoate or L-2-amino-3-(guanidinooxy)propionate, the higher or lower homolog of L-canavanine, respectively. L-Canavanine derivatives such as methyl-L-canavanine, or L-canaline and O-ureido-L-homoserine, are not metabolized significantly by CH.

Acetone↗

PubMed1997

L-canaline: a potent antimetabolite and anti-cancer agent from leguminous plants.

L-Canaline, the L-2-amino-4-(aminooxy)butyric acid structural analog of L-ornithine' is a powerful antimetabolite stored in many leguminous plants. This nonprotein amino acid reacts vigorously with the pyridoxal phosphate moiety of vitamin B6-containing enzymes to form a covalently-bound oxime that inactivates, often irreversibly, the enzyme. Canaline is not only capable of inhibiting ornithine-dependent enzymic activity, but it also can function as a lysine antagonist. Recently, this natural product was found to possess significant antineoplastic in vitro activity against human pancreatic cancer cells.

Aminobutyrates↗

PubMed1996

L-homoarginine studies provide insight into the antimetabolic properties of L-canavanine.

A method for the chemical synthesis of L-homoarginine, based on the guanidination of L-lysine with O-methylisourea, has been developed; this procedure provides radiochemically pure L-[guanidino-14C]homoarginine in high yield. Radiolabeled homoarginine is incorporated readily into the newly synthesized hemolymphic proteins of larvae of the tobacco hornworm, Manduca sexta without adversely affecting larval growth and development. This finding stands in sharp contrast to the effect of L-canavanine, another L-arginine analog, which is markedly deleterious to these larvae. Homoarginine is incorporated into M. sexta lysozyme, and the antibacterial proteins of the fly, Phormia terranovae with impunity. In contrast, the comparable canavanine-containing enzymes are inhibited severely. Experimental evidence is presented that the innocuous nature of homoarginine results from the elevated pKa value of its guanidino group which arguably exceeds even that of arginine. As a result, homoarginine does not disrupt essential residue interactions. In contrast canavanine, which is much less basic than arginine, does adversely affect R group interactions forming the requisite three-dimensional conformation of the protein.

Animals↗

PubMed1996

Structure-activity studies of L-canaline-mediated inhibition of porcine alanine aminotransferase.

L-Canaline [L-2-amino-4-(aminooxy)butanoic acid] (L-CAN) and a family of eleven structurally related analogs were synthesized and evaluated for their inhibitory effect on PLP-dependent alanine aminotransferase (AlaAT) (EC 2.6.1.2) obtained from porcine heart. These congeners were selected to determine the stereochemical, aliphatic chain length, and aminooxy substitutional effects on L-CAN-mediated inhibition of AlaAT activity. L-CAN was the most effective inhibitor of the tested compounds; 10(-7) M L-CAN elicited a 55% reduction in AlaAT activity after a 5 min exposure. This deleterious effect results from the ability of L-CAN to react avidly with PLP moiety of the enzyme to form a stable, L-CAN-PLP oxime. In contrast, the methyl and ethyl esters of L-CAN reduced AlaAT activity by only 8% and 6%, respectively. While all of the L-enantiomeric forms of the tested compound were more potent AlaAT inhibitors than their corresponding D-stereoisomers, the D-enantiomers, particularly D-canaline, were active. Chain shortening or lengthening dramatically curtailed L-CAN-mediated loss in AlaAT activity, but the replacement of the alpha-amino group with a hydrogen was of little consequence in this regard. AlaAT was treated with L-CAN in the presence of free PLP to assess PLP capacity to protect AlaAT against 10(7) M L-CAN-dependent inactivation. L-CAN retained approximately two-thirds of its inhibitory ability in the presence of equimolar PLP, but AlaAT inhibition was reduced 90% by a 10-fold excess of PLP over L-CAN.

Alanine Transaminase↗

PubMed1995

Combination therapy with 5-fluorouracil and L-canavanine: in vitro and in vivo studies.

L-Canavanine (CAV) is a potent L-arginine antagonist, produced by legumes such as the jack bean, Canavalia ensiformis. CAV is cytotoxic to MIA PaCa-2 human pancreatic cancer cells. We sought to determine whether CAV's efficacy as an anticancer agent might be increased in combination with 5-fluorouracil (5-FU), a pyrimidine antimetabolite with activity against solid tumors. Using optimal conditions for the expression of CAV's cytotoxicity against MIA PaCa-2 cells, CAV was more cytotoxic to the cells than 5-FU. The combination of both drugs at a fixed molar ratio of 1:1 exhibited synergistic effects in the cells as determined by combination index analysis. The combination of 5-FU:CAV was tested at a ratio of 5:1 and exhibited antagonism at lower effect levels, additivity at 50% effect levels and slight synergism at higher effect levels. A 10:1 combination of both drugs (5-FU:CAV) exhibited antagonistic effects at all levels. When the drugs were combined at a molar ratio of 20:1, increased antagonism was observed. When CAV (1.0 or 2.0 g/kg daily) and/or 5-FU (35 mg/kg daily) was administered to colonic tumor-bearing rats for five consecutive days, the antitumor activity of the drug combination was significantly greater than the combined effects of either drug alone. However, the body weight loss experienced by CAV-treated rats was increased in those rats exposed to a combination of both drugs. These studies using different tumors provide in vitro and in vivo evidence that combination therapy offers a viable means of improving CAV's intrinsic efficacy while decreasing the concentration of 5-FU required to produce the same cytotoxic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite L-canavanine.

L-Canavanine (CAV), the L-2-amino-4-guanidinooxy structural analogue of L-arginine (ARG), is a potent ARG antagonist which occurs in the jack bean, Canavalia ensiformis. This ARG antimetabolite is active against L1210 murine leukemia and a solid colonic tumor in the rat. Our initial studies using a microtiter assay show that CAV exhibits a 50% inhibitory concentration of approximately 2 mM against the human pancreatic adenocarcinoma cell line, MIA PaCa-2, when these cells are grown in Dulbecco's modified Eagle's medium containing 0.4 mM ARG. When the ARG concentration is reduced to 0.4 microM, the 50% inhibitory concentration for CAV falls precipitously to 0.01 mM. The pronounced increase in the ability of CAV to inhibit MIA PaCa-2 cell growth at the lower ARG concentration may result from enhanced CAV competition with ARG for incorporation into newly synthesized cellular proteins. At 0.4 microM ARG, 30 mM CAV almost completely inhibits cell growth by 6 h. In contrast, with 0.4 mM ARG, complete inhibition does not occur until after 48 h. A dramatic reversal of growth inhibition caused by a very high concentration of CAV was observed when cells treated with CAV were replenished with a high concentration of ARG. Our results suggest that CAV has real potential as a lead compound for the development of analogues with enhanced activity against human pancreatic cancer.

Arginine↗

PubMed1992

Purification and characterization of the higher plant enzyme L-canaline reductase.

A newly discovered enzyme, L-canaline reductase (NADPH:L-canaline oxidoreductase, EC 1.6.6-), has been isolated and purified from 10-day-old leaves of the jack bean Canavalia ensiformis (Leguminosae). This higher plant is representative of a large number of legumes that synthesize L-canavanine, an important nitrogen-storing nonprotein amino acid. Canavanine-storing legumes contain arginase, which hydrolyzes L-canavanine to form the toxic metabolite L-canaline. Canaline reductase, having a mass of approximately 167 kDa and composed of 82-kDa dimers, catalyzes a NADPH-dependent reductive cleavage of L-canaline to L-homoserine and ammonia. This is the only enzyme known to use reduced NADP to cleave an O-N bond. Canaline reductase performs at least three important functions for canavanine-synthesizing legumes. First, it detoxifies canaline. Second, it increases by one-half the overall yield of ammoniacal nitrogen released from canavanine. Third, it permits the carbon skeleton of canavanine, a secondary plant metabolite, to support vital primary metabolic reactions.

Amino Acids↗

PubMed1991

Studies of L-canavanine incorporation into insectan lysozyme.

L-Canavanine is incorporated into the lysozyme synthesized, in response to administration of bacterial cell wall materials, by canavanine-treated larvae of the tobacco hornworm Manduca sexta (Sphingidae). Maximum canavanine incorporation into M. sexta lysozyme occurs when the larvae are provided 1 mg of canavanine g-1 fresh body weight. Analysis of canavanine-containing lysozyme purified from these insects reveals that 21% of the arginine residues are replaced by canavanine; this residue substitution results in a loss of 49.5% of the catalytic activity. When the larvae are provided 0.5 mg of canavanine g-1, 16.5% of the arginine residues are substituted by canavanine and 39.5% of the catalytic activity is lost. Canavanine is also incorporated into the lysozyme induced by canavanine-treated pupae of the giant silk moth Hyalophora cecropia (Saturnidae). In contrast, replacement of 17% of the arginine in H. cecropia lysozyme by canavanine fails to affect the catalytic activity. We have determined the primary structure of M. sexta lysozyme and compared it with the primary structure of H. cecropia lysozyme which has been described elsewhere. M. sexta lysozyme has an arginine at positions 23, 42, and 107. H. cecropia contains serine, lysine, and lysine, respectively, at these locations. The ability of incorporated canavanine to inhibit M. sexta lysozyme activity selectively may result from the fact that replacement of any one of the 3 arginine residues at position 23, 42, or 107 by canavanine causes the loss of catalytic activity.

Amino Acid Sequence↗

PubMed1990

Interaction of L-canaline with ornithine aminotransferase of the tobacco hornworm, Manduca sexta (Sphingidae).

Ornithine aminotransferase (L-ornithine:2-oxo-acid aminotransferase (EC 2.6.1.13)) has been purified to homogeneity from last instar larvae of the tobacco hornworm, Manduca sexta (Sphingidae). This enzyme is a 144,000-Da tetramer constructed from 36,000-Da protomeric units. It has a high aspartate/asparagine and glutamate/glutamine content and 2 cysteine residues/subunit. All 8 cysteine residues can react with N-ethylmaleimide to inactivate the enzyme. Maintenance of the enzyme in the presence of 2-mercaptoethanol and dithiothreitol maximizes enzymatic activity and improves storage conditions, presumably by protecting these sulfhydryl groups. The apparent Km values for L-ornithine and 2-oxoglutaric acid are 2.3 and 3.2 mM, respectively. The turnover number is 2.0 +/- 0.1 mumol min-1 mumol-1. L-Canaline (L-2-amino-4-(aminooxy)butyric acid) is a potent ornithine aminotransferase inhibitor. Reaction of the enzyme with L-[U-14C]canaline produces an enzyme-bound, covalently linked, radiolabeled canaline-pyridoxal phosphate oxime. The L-[U-14C]canaline-pyridoxal phosphate oxime has been isolated from canaline-treated enzyme. Dialysis of canaline-inactivated ornithine aminotransferase against free pyridoxal phosphate slowly reactivates the enzyme as the oxime is replaced by pyridoxal phosphate. Analysis of L-[U-14C]canaline binding to ornithine aminotransferase reveals the presence of 4 mol of pyridoxal phosphate/mol of enzyme.

Amino Acids↗

PubMed1990

Metabolism of l-Canavanine and l-Canaline in Leguminous Plants.

Massive accumulation of l-canavanine, the 2-amino-4-(guanidinooxy)butyric acid structural analog of l-arginine, occurs in many legumes. Accumulation of large amounts of this nonprotein amino acid results in large part from canavanine's protective efficacy; it forms an effective chemical barrier to predation, disease, and even competition with other plants. Diversion of metabolic resources for the synthesis and storage of appreciable canavanine does not place an inordinate burden on the plant. Catabolism of this nonprotein amino acid provides respiratory carbon, generates essential primary metabolites, and ammoniacal nitrogen for the developing plant.

Journal Article↗

PubMed1990

A higher plant enzyme exhibiting broad acceptance of stereoisomers.

An arginase, purified from the leaf of the jack bean, Canavalia ensiformis, can effectively hydrolyze both l- and d-arginine. Arginases, examined from a number of other plant and animal sources, exhibit marked substrate stereospecificity and fail to catabolize d-arginine. In order to provide essential nitrogen, jack bean leaf arginase also catabolizes l-canavanine, an arginine analog that is a predominant nitrogen-storing metabolite of this legume. The ability of arginase to metabolize both stereoisomers of arginine may result from the requirement for this enzyme to exhibit limited substrate specificity in order to hydrolyze both arginine and canavanine.

Journal Article↗

PubMed1989

L-canavanine incorporation into vitellogenin and macromolecular conformation.

L-Canavanine is a potentially deleterious arginine antimetabolite whose toxicity is expressed in canavanine-sensitive organisms ranging from viruses to humans. Canavanine, a substrate for arginyl-tRNA synthetase, is incorporated into nascent polypeptide chains in place of arginine. This substitution results in the production of structurally aberrant, canavanyl proteins. Chemical, physical, and immunological studies of native and canavanine-containing vitellogenin obtained from female migratory locusts (Locusta migratoria migratorioides (Orthoptera] provide the first experimental evidence that canavanine can disrupt the tertiary and/or quaternary structure that yields the three-dimensional conformation unique to the protein. These findings enhance our understanding of the biochemical basis for canavanine's antimetabolic and potent insecticidal properties.

Animals↗

PubMed1989

Canavanine incorporation into the antibacterial proteins of the fly, Phormia terranovae (Diptera), and its effect on biological activity.

In response to microbial infection or mechanical injury, larvae of the fly, Phormia terranovae (Diptera), can induce de novo production of a group of antibacterial proteins including: peak I protein, diptericin A, diptericin B, diptericin C, and peak V protein. Administration of L-canavanine at the time of mechanical injury results in the incorporation of this arginine antagonist into these proteins. Canavanine replacement for arginine causes a total loss of detectable antibacterial activity for diptericin B and diptericin C, whereas diptericin A and peak V protein are severely inhibited. This loss in biological activity occurs in spite of the fact that canavanine stimulates induced protein synthesis. Analysis of the hydrolysate of diptericin A reveals that one-third of the 3 arginyl residues are replaced by canavanine. This investigation provides the first evidence that canavanine incorporation into a protein can impair its function.

Amino Acids↗

PubMed1989

Induction of macrophage procoagulant activity by Bacteroides fragilis.

Fibrin deposition in the peritoneal cavity during acute peritonitis appears to predispose the host to abscess formation by providing an environment for bacterial proliferation protected from host defenses. The purpose of the present study was to determine whether the potent abscess-inducing anaerobe Bacteroides fragilis could promote fibrin deposition by inducing mononuclear cells to express procoagulant activity (PCA). B. fragilis stimulated PCA in a dose-dependent fashion, achieving a maximum at 10(7) CFU/ml. Heat-killed B. fragilis induced comparable levels of PCA, while a nonspecific phagocytic stimulus, latex beads, was not stimulatory. B. fragilis was capable of inducing PCA even when phagocytosis was blocked by preexposure of cells to latex beads. The results suggested that phagocytosis was neither necessary nor sufficient for the generation of PCA. Cell separation studies showed that PCA was solely produced by macrophages and that lymphocytes did not augment its production. These studies suggest one potential mechanism by which B. fragilis might initiate abscess formation.

Animals↗

PubMed1988

Prevention of intra-abdominal abscesses with fibrinolytic agents.

Fibrin deposition during secondary peritonitis predisposes to abscess formation by protecting bacteria from host-defence mechanisms. To test the hypothesis that local fibrinolytic therapy can prevent the formation of intra-abdominal abscess, daily injections of the fibrinolytic enzymes trypsin and tissue plasminogen activator (t-PA) were administered intraperitoneally to Wistar rats inoculated intraperitoneally with infected fibrin clots. After 5 days, trypsin (1 mg/ml) had significantly (p less than 0.001) reduced abscess formation in animals inoculated with monomicrobial Bacteroides fragilis clots (20% versus 87%) or mixed Escherichia coli-B. fragilis clots (11% versus 91%). Bacteroides fragilis abscesses were also completely prevented with t-PA (0.25 mg/ml). The number of B. fragilis organisms present in residual abscesses in the trypsin-treated group was significantly (p less than 0.05) lower than in the control group (8.2 +/- 0.2, n = 7 versus 5.7 +/- 1.4, n = 4, log CFU/g abscess). In-vitro studies demonstrated that trypsin had no bactericidal effect on B. fragilis, suggesting enhanced clearance of bacteria. From these studies it appears that controlled fibrinolysis at operation may be a useful adjunct to surgery and systemic antibiotics in preventing abscess formation postoperatively.

Abdomen↗

PubMed1987

Aberrant, canavanyl protein formation and the ability to tolerate or utilize L-canavanine.

L-Canavanine, 2-amino-4-(guanidinooxy)butyric acid, and L-arginine incorporation into de novo synthesized proteins was compared in six organisms. Utilizing L-[guanidinooxy14C]canavanine and L-[guanidino14C]arginine at substrate saturation, the canavanine to arginine incorporation ratio was determined in de novo synthesized proteins. Caryedes brasiliensis and Sternechus tuberculatus, canavanine utilizing insects; Canavalia ensiformis, a canavanine storing plant; and to a lesser extent Heliothis virescens, a canavanine resistant insect, failed to accumulate significant canavanyl proteins. By contrast, Manduca sexta, a canavanine-sensitive insect, and Glycine max, a canavanine free plant, readily incorporated canavanine into newly synthesized proteins. This study supports the contention that the incorporation of canavanine into proteins in place of arginine contributes significantly to canavanine's antimetabolic properties.

Animals↗