Germination of Witchweed (Striga lutea Lour.): Isolation and Properties of a Potent Stimulant.

A crystalline germination stimulant (trivial name strigol) for the rootparasite, witchweed (Striga lutea Lour.), has been isolated from cotton rootexudates and characterized as a C(19)H(22)O(6) compound. Although apparently different from known plant hormones, the stimulant is active at hormonal levels, causing germination at concentrations less than 1O(-5) part per million.

Intravenous injection in man of 9 -tetrahydrocannabinol and 11-OH- 9 -tetrahydrocannabinol.

A microsuspension of Delta(9)-tetrahydrocannabinol and of its metabolic derivative 11-OH-Delta(9)-tetrahydrocannabinol has been prepared with 25 percent human serum albumin as the vehicle. Intravenous infusion of this preparation to humans indicates that both tetrahydrocannabinols are equally potent in producing the typical marihuana-like pschological and physiological effects.

Activity of delta8- and delta9-tetrahydrocannabinol and related compounds in the mouse.

The 11-hydroxy metabolites of Delta(8).- and Delta(9)-tetrahydrocannabinol are more active than the parent compounds when administered to mice by either the intravenous or intracerebral route. Both Delta(8)- and Delta(9)-tetrahydrocannabinol are rapidly and extensively metabolized by the liver and not by the brain. The hypothesis that the 11-hydroxy metabolites may be the active form of tetrahydrocannabinol is discussed

DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts.

Drug development is needed to improve chemotherapy of patients with locally advanced or metastatic colon carcinoma, who otherwise have an unfavorable prognosis. DNA topoisomerase I, a nuclear enzyme important for solving topological problems arising during DNA replication and for other cellular functions, has been identified as a principal target of a plant alkaloid 20(S)-camptothecin. Significantly increased concentrations of this enzyme, compared to that in normal colonic mucosa, were found in advanced stages of human colon adenocarcinoma and in xenografts of colon cancer carried by immunodeficient mice. Several synthetic analogs of camptothecin, selected by tests with the purified enzyme and tissue-culture screens, were evaluated in the xenograft model. Unlike other anticancer drugs tested, 20(RS)-9-amino-camptothecin (9-AC) induced disease-free remissions. The overall drug toxicity was low and allowed for repeated courses of treatment.

Motions of calmodulin characterized using both Bragg and diffuse X-ray scattering.

BACKGROUND: Calmodulin is a calcium-activated regulatory protein which can bind to many different targets. The protein resembles a highly flexible dumbbell, and bends in the middle as it binds. This and other motions must be understood to formulate a realistic model of calmodulin function. RESULTS: Using the Bragg reflections from X-ray crystallography, a multiple-conformer refinement of a calmodulin-peptide complex shows anisotropic displacements, with high variations of dihedral angles in several nonhelical domains: the flexible linker; three of the four calcium-binding sites (including both of the N-terminal sites); and a turn connecting the C-terminal EF-hand calcium-binding domains. Three-dimensional maps of the large scale diffuse X-ray scattering data show isotropic liquid-like motions with an unusually small correlation length. Three-dimensional maps of the small scale diffuse streaks show highly coupled, anisotropic motions along the head-to-tail molecular packing direction in the unit cell. There is also weak coupling perpendicular to the head-to-tail packing direction, particularly across a cavity occupied by the disordered linker domain of the molecule. CONCLUSIONS: Together, the Bragg and diffuse scattering present a self-consistent description of the motions in the flexible linker of calmodulin. The other mobile regions of the protein are also of great interest. In particular, the high variations in the calcium-binding sites are likely to influence how strongly they bind ions. This is especially important in the N-terminal sites, which regulate the activity of the molecule.

High-resolution macromolecular structure determination using CCD detectors and synchrotron radiation.

BACKGROUND: Synchrotron radiation sources have made impressive contributions to macromolecular crystallography. The delay in development of appropriate X-ray detectors has, however, been a significant limitation to their efficient use. New technologies, based on charge-coupled devices (CCDs), provide capabilities for faster, more accurate, automated data collection. RESULTS: A CCD-based X-ray detector has been developed for use in macromolecular crystallography and has been in operation for about one and a half years at the Cornell High Energy Synchrotron Source. It has been used for a variety of crystallographic projects, including a number of high-resolution structural studies. The statistical quality of the data, the detector's ease and efficiency of use, and the growing number of structural results illustrate the practical utility of this new detector system. CONCLUSIONS: The new detector has enhanced capabilities for measuring diffraction patterns from crystals of macromolecules, especially at high resolution, when the X-ray intensities are weak. The survey of results described here ranges from virus crystallography to weakly diffracting small-molecule structure determination and demonstrates the potential of CCD detectors when combined with synchrotron radiation sources.

Camptothecin and taxol: discovery to clinic--thirteenth Bruce F. Cain Memorial Award Lecture.

Camptothecin and taxol are secondary metabolites found, respectively, in the wood bark of Camptotheca acuminata, a native of China, and Taxus brevifolia, found in the northwest Pacific coastal region of the United States. The compounds were isolated guided by bioassay on various extracts and chromatographic fractions. Their unique and hitherto unknown structures were elucidated by nuclear magnetic resonance, mass spectrometry, and X-ray analysis. Both compounds have unique mechanisms of antitumor activity; camptothecin uniquely inhibits an enzyme, topoisomerase I, involved in DNA replication. Taxol binds to a protein, tubulin, thus inhibiting cell division. Taxol has been called the best new anticancer agent developed from natural products, showing particular efficacy against ovarian cancer. Camptothecin and analogues singly or combined with cisplatin show efficacy against solid tumors, breast, lung, and colorectal, which hitherto have been unaffected by most cancer chemotherapeutic agents.

Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells.

In order to understand the high efficacy of camptothecin derivatives against human colon tumor xenografts in nude mice, we have studied the transport properties of camptothecin derivatives across cellular membranes of MDR1-overexpressing cells. MDR1 overexpression was shown to have little effect on camptothecin cytotoxicity; camptothecin was equally cytotoxic to both the drug-sensitive parental cell line, KB 3-1, and its multidrug-resistant derivative, KB V1. The ability of camptothecin to overcome MDR1-mediated resistance is most likely due to unimpaired accumulation of camptothecin in MDR1 cells as suggested from the following experiments: (a) cytotoxicity of camptothecin against KB V1 cells was not altered by the known MDR1-reversing agent, verapamil; (b) camptothecin was ineffective as compared with vinblastine in competing with [3H]azidopine for photoaffinity labeling of MDR1; (c) camptothecin was equally efficient in trapping cellular topoisomerase I molecules on chromosomal DNA in the form of cleavable complexes in both KB 3-1 and KB V1 cells. The mechanism by which camptothecin overcomes MDR1-mediated resistance has been further studied using a number of uncharged and charged camptothecin derivatives. In contrast to the uncharged camptothecin derivatives, such as 9-amino-camptothecin and 10,11-methylenedioxy-camptothecin, the charged camptothecin derivative, topotecan, showed reduced cytotoxicity against MDR1-overexpressing KB V1 cells. The reduced cytotoxicity of topotecan in KB V1 cells was due to the overexpression of MDR1 in KB V1 cells since verapamil restored both topotecan accumulation and cytotoxicity. These results suggest that the charge on camptothecin can affect the drug's sensitivity to MDR1. The possible effect of membrane permeability in determining drug selectivity of MDR1 is discussed.

Structure-activity study of the actions of camptothecin derivatives on mammalian topoisomerase I: evidence for a specific receptor site and a relation to antitumor activity.

Twenty-two compounds related to camptothecin, a known inhibitor of eukaryotic topoisomerase I, were studied. The following effects on the actions of topoisomerase I were observed and were well correlated among most of the compounds studied: (a) inhibition of the first-order rate of relaxation of supercoiled DNA; (b) conversion of supercoiled DNA to nicked circles; and (c) single-strand cleavage of linear DNA at specific sites. The locations of the stimulated cleavage sites were the same for all of the active derivatives. Stereochemistry and the positions of substituents were found to be crucial for the presence or absence of effects on topoisomerase I, indicating that the compounds interact with an asymmetrical receptor site on the enzyme or enzyme-DNA complex. From the structure-activity relations, the regions of interaction between the camptothecin ring system and the receptor site were inferred. Striking correlations were observed between activity against topoisomerase I and reported activity against murine leukemias, indicating that an action on topoisomerase I is responsible for the antitumor activity of the camptothecins.

Water soluble 20(S)-glycinate esters of 10,11-methylenedioxycamptothecins are highly active against human breast cancer xenografts.

Water-soluble 20(S)-glycinate esters of two highly potent 10,11-methylenedioxy analogues of camptothecin (CPT) have been synthesized and evaluated for their ability to eradicate human breast cancer tumor xenografts. The glycinate ester moiety increases the water solubility of the 10,11-methylenedioxy analogues 4-16-fold. However, in contrast to CPT-11, a water-soluble CPT analogue that was recently approved for second line treatment of colorectal cancer, the 20(S)-glycinate esters do not require carboxylesterase for conversion to their active forms. The glycinate esters are hydrolyzed to their parent, free 20(S)-hydroxyl active analogues in phosphate buffer (pH 7.5) and in mouse and human plasma. The glycinate esters are also 20-40-fold less potent than CPT-11 in inhibiting human acetylcholinesterase. In vivo, we examined 20(S)-glycinate-10,11-methylenedioxycamptothecin, 20(S)-glycinate-7-chloromethyl-10,11-methylenedioxycamptothecin, and CPT-11. We found that the two 10,11-methylenedioxy analogues had antitumor activity against breast cancer xenografts that was comparable to that of CPT-11. Our results indicate that water-soluble 20(S)-glycinate esters of highly potent CPT analogues provide compounds that maintain biological activity, do not require interactions with carboxylesterases, and do not inhibit human acetylcholinesterase.

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues.

20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.

Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication.

Tendon and other connective tissue cells are subjected to diverse mechanical loads during daily activities. Thus, fluid flow, strain, shear and combinations of these stimuli activate mechanotransduction pathways that modulate tissue maintenance, repair and pathology. Early mechanotransduction events include calcium (Ca2+) signaling and intercellular communication. These responses are mediated through multiple mechanisms involving stretch-activated channels, voltage-activated channels such as Ca(v)1, purinoceptors, adrenoceptors, ryanodine receptor-mediated Ca2+ release, gap junctions and connexin hemichannels. Calcium, diacylglycerol, inositol (1,4,5)-trisphosphate, nucleotides and nucleosides play intracellular and/or extracellular signaling roles in these pathways. In addition, responses to mechanical loads in tendon cells vary among species, tendon type, anatomic location, loading conditions and other factors. This review includes a synopsis of the immediate responses to mechanical loading in connective tissue cells, particularly tenocytes. These responses involve Ca2+ signaling, gap junctions and intercellular communication.

Dual role of glutathione in modulating camptothecin activity: depletion potentiates activity, but conjugation enhances the stability of the topoisomerase I-DNA cleavage complex.

Depletion of glutathione (GSH) in MCF-7 and MDA-MB-231 cell lines by pretreatment with the GSH synthesis inhibitor buthionine sulfoximine potentiated the activity of 10,11-methylenedioxy-20(S)-camptothecin, SN-38 [7-ethyl-10-hydroxy-20(S)-camptothecin], topotecan, and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC). The greatest potentiation was observed with the alkylating camptothecin CMMDC. Buthionine sulfoximine pretreatment also increased the number of camptothecin-induced DNA-protein crosslinks, indicating that GSH affects the mechanism of action of camptothecin. We also report that GSH interacts with CMMDC to form a stable conjugate, 7-(glutathionylmethyl)-10,11-methylenedioxy-20(S)-camptothecin (GSMMDC), which is formed spontaneously in buffered solutions and in MCF-7 cells treated with CMMDC. GSMMDC was synthesized and found to be nearly as active as 10,11-methylenedioxy-20(S)-camptothecin in a topoisomerase (topo) I-mediated DNA nicking assay. The resulting topo I cleavage complexes were remarkably stable. In cell culture, GSMMDC displayed potent growth-inhibitory activity against U937 and P388 leukemia cell lines. GSMMDC was not active against a topo I-deficient P388 cell line, indicating that topo I is its cellular target. Peptide-truncated analogues of GSMMDC were prepared and evaluated. All three derivatives [7-(gamma-glutamylcysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, 7-(cysteinylglycylmethyl)-10,11-methylenedioxy-20(S)-camptothecin, and 7-(cysteinylmethyl)-10,11-methylenedioxy-20(S)-camptothecin] displayed topo I and cell growth-inhibitory activity. These results suggest that 7-peptidyl derivatives represent a new class of camptothecin analogues.

Efficient parallel linear scaling construction of the density matrix for Born-Oppenheimer molecular dynamics.

We present an algorithm for the calculation of the density matrix that for insulators scales linearly with system size and parallelizes efficiently on multicore, shared memory platforms with small and controllable numerical errors. The algorithm is based on an implementation of the second-order spectral projection (SP2) algorithm [ Niklasson, A. M. N. Phys. Rev. B 2002 , 66 , 155115 ] in sparse matrix algebra with the ELLPACK-R data format. We illustrate the performance of the algorithm within self-consistent tight binding theory by total energy calculations of gas phase poly(ethylene) molecules and periodic liquid water systems containing up to 15,000 atoms on up to 16 CPU cores. We consider algorithm-specific performance aspects, such as local vs nonlocal memory access and the degree of matrix sparsity. Comparisons to sparse matrix algebra implementations using off-the-shelf libraries on multicore CPUs, graphics processing units (GPUs), and the Intel many integrated core (MIC) architecture are also presented. The accuracy and stability of the algorithm are illustrated with long duration Born-Oppenheimer molecular dynamics simulations of 1000 water molecules and a 303 atom Trp cage protein solvated by 2682 water molecules.

Protein structure determination using a database of interatomic distance probabilities.

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.

A model of troponin-I in complex with troponin-C using hybrid experimental data: the inhibitory region is a beta-hairpin.

We present a model for the skeletal muscle troponin-C (TnC)/troponin-I (TnI) interaction, a critical molecular switch that is responsible for calcium-dependent regulation of the contractile mechanism. Despite concerted efforts by multiple groups for more than a decade, attempts to crystallize troponin-C in complex with troponin-I, or in the ternary troponin-complex, have not yet delivered a high-resolution structure. Many groups have pursued different experimental strategies, such as X-ray crystallography, NMR, small-angle scattering, chemical cross-linking, and fluorescent resonance energy transfer (FRET) to gain insights into the nature of the TnC/TnI interaction. We have integrated the results of these experiments to develop a model of the TnC/TnI interaction, using an atomic model of TnC as a scaffold. The TnI sequence was fit to each of two alternate neutron scattering envelopes: one that winds about TnC in a left-handed sense (Model L), and another that winds about TnC in a right-handed sense (Model R). Information from crystallography and NMR experiments was used to define segments of the models. Tests show that both models are consistent with available cross-linking and FRET data. The inhibitory region TnI(95-114) is modeled as a flexible beta-hairpin, and in both models it is localized to the same region on the central helix of TnC. The sequence of the inhibitory region is similar to that of a beta-hairpin region of the actin-binding protein profilin. This similarity supports our model and suggests the possibility of using an available profilin/actin crystal structure to model the TnI/actin interaction. We propose that the beta-hairpin is an important structural motif that communicates the Ca2+-activated troponin regulatory signal to actin.

Metabolism, disposition, and kinetics of delta-9-tetrahydrocannabinol in men and women.

A comparative study was done in women and men of the effects of delta 9-tetrahydrocannabinol (delta 9-THC), intravenously or orally, on dynamic activity, metabolism, excretion, and kinetics. In general no differences between the two sexes were observed. delta 9-THC is converted by microsomal hydroxylation to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC), which is both a key intermediate for further metabolism to 11-nor-delta 9-THC-9-carboxylic acid (11-nor-acid) by liver alcohol-dehydrogenase enzymes and a potent psychoactive metabolite. Major differences in the ratio of the concentration of 11-OH-delta 9-THC to that of delta 9-THC in plasma were found after intravenous dosing (ratio 1:10 to 20) compared with oral administration (ratio 0.5 to 1:1). The final metabolic products are the 11-nor-acids and the related, more polar acids. Urinary excretion of delta 9-THC is restricted to acidic nonconjugated and conjugated metabolites. After 72 hr mean cumulative urinary excretion, noted for both routes and for both sexes, ranged from 13% to 17% of the total dose. After 72 hr the cumulative fecal excretion for both sexes after intravenous administration ranged from 25% to 30%; after oral administration the range was 48% to 53%. Metabolites were found in the feces in large concentration in the nonconjugated form; concentrations of 11-OH-delta 9-THC were particularly noteworthy. Kinetics of delta 9-THC and metabolites were much the same for female and male subjects. For delta 9-THC, terminal-phase t1/2s for both sexes, irrespective of the route, ranged from 25 to 36 hr. A comparison of the results for AUC/dose (delta 9-THC) after oral dosing with comparable data from intravenous administration indicated bioavailability of the order of 10% to 20% for both sexes. After intravenous delta 9-THC, large apparent volumes of distribution were noted (about 10 l/kg for both sexes).

A complex pattern of disposition of phenytoin in severe intoxication.

A 5-year-old child developed phenytoin (diphenylhydantoin, DPH) toxicity after receiving 500 mg of the drug daily for 3 weeks. Plasma, urine, and duodenal fluid were collected for assay of DPH and its metabolites. The peak plasma concentration of DPH was 108 mug/ml, and the decline in plasma level did not fit first-order kinetics. The para-hydroxy, meta-hydroxy, and dihydrodiol metabolites of DPH were measured in urine; duodenal aspirate contained both DPH and the para-hydroxy metabolite. Plasma pH may affect distribution of DPH since in vitro binding of DPH to human albumin increased as pH increased.

Phencyclidine disposition after intravenous and oral doses.

[3H]-Phencyclidine (PCP) hydrochloride was given in intravenous (0.1 or 1 mg) or oral (1 mg) doses to male subjects. After 1 mg IV, drug and metabolites were recovered in urine (72.8 +/- 4.0% of dose), feces (4.7 +/- 0.9%), and perspiration. Fecal excretion was low (3.4 +/- 0.4%) after oral dosing and oral bioavailability was estimated at 72%. PCP comprised 16% of urinary radioactivity with 31% consisting of enzymatically hydrolyzable conjugates of hydroxylated metabolites. Both cis and trans isomers of 4-phenyl-4-(1-piperidinyl)cyclohexanol were found. Maximum average plasma PCP concentrations of 2.7 to 2.9 ng/ml were observed after oral and intravenous 1-mg doses. Blood/plasma ratios were approximately 1.0 and plasma binding was about 65%. Parent drug was found in saliva. Apparent terminal phase half-lifes averaged 21 +/- 3 hr (harmonic mean 17 hr, range 7 to 46 hr). The volume of distribution averaged 6.2 +/- 0.3 l/kg. Renal clearances were variable, but the average was 9% of the total clearance. Thus, PCP is cleared principally by metabolism.

Plant antitumor agents. 23. Synthesis and antileukemic activity of camptothecin analogues.

Eight optically active and nine racemic ring A modified analogues of 20(S)-camptothecin were prepared and evaluated for antitumor activity in the L-1210 leukemia system. The ring A mono- and disubstituted analogues displayed a wide variance in activity and potency. It was found that monosubstitution by NH2 or OH at positions 9, 10, or 11 yielded compounds with activity much higher than the parent compound, camptothecin, whereas substitution at position 12 greatly reduced activity. In general, disubstitution in ring A greatly reduced antileukemic activity. Replacement of ring A by heterocyclic rings (thiophene or pyridine) leads to analogues with only moderate activity.