Naturally Occurring Cardenolides Affecting Schistosoma mansoni.

Schistosomiasis is a neglected tropical disease of considerable public health burden. We recently discovered a micromolar activity of several cardenolides against newly transformed schistosomula (NTS) of the parasitic flatworm Schistosoma mansoni in a small compound screen including different substance classes of both natural products as well as synthetic molecules. In further experiments, a focused library of naturally occurring and synthetic steroids was explored against NTS and adult S. mansoni, revealing seven cardenolides with comparable activities as known anthelminthics such as praziquantel. Of these, gomphoside monoacetate and uscharin showed suitable therapeutic indices. In a first in vivo study, at a dose of 10 mg/kg, only minor activity in mice harboring a chronic S. mansoni infection could be shown, which will be further investigated by structure-activity relationship studies as well as pharmacodynamic and pharmacokinetic approaches.

Interactions of a herbal combination that inhibits growth of prostate cancer cells.

PURPOSE: PC SPES is an eight-component herbal product marketed for the treatment of prostate cancer. The manufacturer of PC SPES claims that the herbal combination is a synergistic blend, but the purported synergy has never been tested. We examined the interaction in cell culture of these eight individual herbal components by the use of an isobologram. METHODS: US patent no. 5,665,393 (1997) for PC SPES was acquired, and each of the eight herbal components described was acquired, properly identified, and extracted by 95% ethanol. The extracts were tested for cytotoxicity to PC 3 human prostate cancer cells in culture by the MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Seven combinations of herbal extracts were made, varying in the proportion of the most cytotoxic herbal extract, that of Panax notoginseng. The interactions of P. notoginseng with the other seven herbs were evaluated through the use of an isobologram. RESULTS: In all seven herbal combinations, P. notoginseng was found to be antagonistic with the other seven herbal components in the cytotoxicity assay ( P values: 0.09, 0.12, 0.12, 0.33, 0.45, 0.56, and 0.76). CONCLUSIONS: The interaction between the most cytotoxic herbal component of a widely used herbal product and the other seven components was antagonistic. Herbal combinations are no different from traditional combination pharmacotherapy. If herbal combinations are able to achieve antagonism, then theoretically they can achieve synergism if combined properly.

Comparison of bone marrow extracellular matrices.

We have compared the structure and composition of adult and fetal bovine bone marrow extracellular matrices. In contrast to fetal bone marrow, adult bone marrow has more oval fenestration and accumulation of adipocytes as well as lower protein content. These differences could be due to remodeling of bone marrow tissue as it develops. Zymogram analysis of matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) activities showed that fetal, but not adult bone marrow extract contained a 96-kDa MMP and TIMP-1 and -2. These activities may contribute to the structural differences between adult and fetal bone marrow tissues.

Light activates reduction of methotrexate by NADPH in the ternary complex with Escherichia coli dihydrofolate reductase.

Methotrexate (MTX), a strong inhibitor of dihydrofolate reductase (DHFR), has been widely used for chemotherapy for many types of cancer as well as for juvenile rheumatoid arthritis. It mimics folate substrates and binds tightly to the active site of DHFR, perhaps in a conformation close to the transition state of the folate catalyzed reaction. Absorption, fluorescence and ultrasensitive Raman difference spectroscopies show that light-activated MTX reacts with NADPH in the enzyme active site, producing 5,8-dihydromethotrexate (5,8-dihydro-MTX) and NADP+. The reaction, which proceeds with a hydride transfer between C4 (pro-R side) of the nicotinamide ring and N5 of the pteridine ring, is similar to that between folate and NADPH except that the hydride is transferred to C6 in this case. Hence, MTX is catalytically competent in its excited state. Most experiments were performed on the Escherichia coli enzyme, but preliminary studies show that the reaction also occurs with human DHFR.

Degradation products of beclomethasone dipropionate in human plasma.

The anti-inflammatory glucocorticosteroid beclomethasone dipropionate was found previously to degrade in human plasma at 37 degrees C to yield beclomethasone 17-monopropionate, beclomethasone 21-monopropionate, and beclomethasone together with three unknown species, D-1, D-2, and D-3. In this paper, we report the isolation of D-2 and D-3 by preparative HPLC and the elucidation of their structures. Both products D-2 and D-3 exhibited UV bathochromic shifts relative to beclomethasone dipropionate of 9 nm. From the mass spectrometry and 1H-NMR data, it is concluded that D-2 and D-3 are formed from beclomethasone and beclomethasone 21-monopropionate, respectively, with the loss of hydrogen chloride and the formation of a 9,11-epoxide. Data for 1H-NMR methyl chemical shifts are used to show that the epoxide has the mechanistically more plausible 9beta,11beta configuration. Thus, D-2 is 9beta, 11beta-epoxy-16beta-methyl-1,4-pregnadiene-17alpha,21- diol-3, 20-dione, and D-3 is its corresponding 21-propanoate. The various enzyme-catalyzed and nonenzyme-catalyzed reactions involved in the degradation of beclomethasone dipropionate in human plasma are discussed. A degradation scheme is proposed.

The influence of aspartate 26 on the tautomeric forms of folate bound to Lactobacillus casei dihydrofolate reductase.

The ternary complex of Lactobacillus casei dihydrofolate reductase (DHFR) with folate and NADP+ exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pK of approx. 6. To investigate the role of Asp26 in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9-(13)C4]folate in its complex with the mutant DHFR Asp26 --> Asn and NADP+. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4-(13)C]aspartic acid in its complex with folate and NADP+ indicates that no Asp residue has a pK value greater than 5.4. Two of the Asp CO2 signals appear as non-integral signals with chemical shifts typical of non-ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26 influences this indirectly by binding in its CO2 form to the protonated N1 group of folate in forms I and IIa thus reducing the pK involving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.

Isolation and characterization of Caenorhabditis elegans extracellular matrix.

The extracellular matrix (ECM) plays an important structural and functional role in multicellular organisms. Because of the similarities between C. elegans and vertebrate development, this nematode could serve as a simplified model to study the biology of the ECM. In this study, a method for extracting mammalian ECM was adapted for the extraction of C. elegans ECM. ECM components from C. elegans were found to be homologous to mammalian ECM by immunoblotting. It was also demonstrated that antibodies generated against C. elegans ECM stained basement membrane-like structures in C. elegans eggs, larvae, and adults.

Interaction of mouse thymocytes and a thymocyte-like cell line with the ECM glycoprotein entactin.

Entactin, a sulfated glycoprotein of 150-kDa, is a component of the extracellular matrix that promotes the adhesion of numerous types of cells, including lymphocytes (Li and Cheung, J. Immunol. 149, 3174, 1992), prompting us to question whether developing T lymphocytes in the thymus (thymocytes) also interact with this molecule. We thus investigated the adhesion of a thymocyte-like cell line (S49.1) to entactin, as well as the adhesion and migration of primary mouse thymocytes upon entactin-coated surfaces. In dose-response and time-course experiments, a 50 micrograms/mL coating concentration of entactin and a 60-min incubation period induced a high level (approximately 65-85%) of S49.1 cell adhesion. Preincubation of the S49.1 cells in medium containing the metabolic inhibitors sodium azide or 2-deoxy-D-glucose inhibited adhesion to entactin 47.2 and 79.5%, respectively. Furthermore, performing the adhesion assay at 4 degrees C instead of at 37 degrees C inhibited S49.1 cell adhesion 27.1%. A high percentage (approximately 90-100%) of S49.1 cells also bound to the lectin concanavalin A and to fibronectin, while laminin promoted only 19.3% adhesion. Our adhesion assay (St. John et al., J. Immunol. Methods, 170, 159, 1994) was then modified to permit a comparison of S49.1 cell adhesion strength to entactin relative to the other substrates. Consequently, Concanavalin A promoted the strongest adhesion, followed by fibronectin and then entactin. In addition, high percentages (92.5, 63, and 75.9%, respectively) of primary thymocytes from 4- to 5-week-old BALB/c mice adhered to entactin, Con A, and fibronectin, while much lower levels (7.6%) of adhesion to laminin were observed. Using a capillary tube random migration assay to measure haptokinesis, entactin-, concanavalin A-, and fibronectin-coated surfaces stimulated little migration, while laminin-coated surfaces enhanced thymocyte migration extensively. Since entactin promoted thymocyte adhesion but affected migration only marginally, we suggest that this molecule may play a role in thymocyte localization during T cell development.

Activation and cytotoxicity of 2-alpha-aminoacyl prodrugs of methotrexate.

In an effort to improve the selectivity of the anticancer drug methotrexate (MTX), a series of potential prodrugs in which the 2-amino group was acylated with various alpha-amino acids (as well as L-pyroglutamic acid) was synthesized. Such derivatives are anticipated to be hydrolysed to MTX by appropriate aminopeptidases localized (over-expressed naturally or targeted as anti-tumor antibody conjugates) in the vicinity of the tumor. The L-leucyl, L-valyl, L-isoleucyl, D-alanyl and L-pyroglutamyl derivatives were assessed as to their suitability as prodrugs. Except for the L-pyroglutamyl compound, all derivatives decomposed slowly when incubated in phosphate buffer, pH 7.3; the formation of MTX was minimal. No major differences were observed when serum was included in the incubation medium, except for the L-leucyl compound, which was hydrolysed to MTX. The L-leucyl, L-valyl and L-isoleucyl derivatives were hydrolysed readily to MTX by aminopeptidase M (EC 3.4.11.2), while the L-pyroglutamyl and D-alanyl compounds were activated by pyroglutamate aminopeptidase (EC 3.4.19.3) (from Bacillus amyloliquefaciens) and D-aminopeptidase (from Ochrobactrum anthropi), respectively. When tested for inhibition of the target enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3), 2-L-valyl-MTX showed inhibition two orders of magnitude poorer than that given by MTX, in agreement with the expectation that acylation of the 2-amino group reduces binding to DHFR. After treatment of this derivative with aminopeptidase M, the extent of inhibition correlated with the amount of MTX formed. MTX derivatives alone or in combination with the complementary peptidase were tested for cytotoxicity on murine L1210 cells in culture. The above-listed derivatives were considerably less cytotoxic than MTX, except for the L-leucyl derivative which showed considerable cytotoxicity. When the appropriate exogenous peptidase was included, the cytotoxicity of the activated prodrugs approached that of MTX. These results indicate that 2-L-leucyl-MTX is unsuitable as a prodrug since it is activated prematurely by serum enzymes. Although the L-valyl and L-isoleucyl derivatives do not hydrolyse to MTX in serum and are readily activated, they are not ideal prodrugs since they decompose under physiological conditions; the properties of the decomposition product will have a bearing on the ultimate suitability of these compounds. 2-L-Pyroglutamyl-MTX is the best candidate prodrug, showing stability and ready activation by the appropriate aminopeptidase.

Solution structure of bound trimethoprim in its complex with Lactobacillus casei dihydrofolate reductase.

Two- and three-dimensional (2D and 3D) NMR techniques have been used to assign the signals from nearly all of the protons in Lactobacillus casei dihydrofolate reductase (DHFR) (M(r) 18,300) in its 1:1 complex with the antibacterial drug trimethoprim. A sample of uniformly 15N-labeled protein was examined using 3D 15N/1H experiments [nuclear Overhauser, heteronuclear multiple quantum coherence (NOESY-HMQC) and total correlation, heteronuclear multiple quantum coherence (TOCSY-HMQC) experiments]. Twenty-two intermolecular NOEs between trimethoprim and protein protons and four intramolecular NOEs in the ligand have been detected. Some were obtained by using heteronuclear editing and 2D HMQC-NOESY experiments on complexes formed with 15N-and 13C-labeled trimethoprim molecules ([1,3-15N2,2-amino-15N]-and [7-13C,4'-methoxy-13C]trimethoprim) bound to unlabeled protein. The ligand-protein NOEs were used as distance constraints in conjunction with minimum energy and simulated annealing calculations (carried out with X-PLOR) to dock the trimethoprim ligand into dihydrofolate reductase, using as a starting structure the crystal coordinates from a related complex with a similar overall protein structure. The restrained minimum energy calculations and the simulated annealing calculations gave 83 calculated structures with distance violations of < 0.1 A. In all of these, the two aromatic rings of trimethoprim occupied essentially the same region of conformational space in the binding site (RMSD = 0.63 A). The protein residues nearest to the bound trimethoprim were found to be very similar in all of the structures and agreed well with corresponding contact residues observed in the X-ray crystal studies on trimethoprim complexes formed with Escherichia coli and chicken liver DHFRs.

An adhesion assay using minimal shear force to remove nonadherent cells.

A new 96-well microtiter plate based adhesion assay was developed to measure weak cell adhesion. This assay is distinct from other adhesion assays by the procedure in which the nonadherent cells are removed. In most conventional adhesion assays, nonadherent cells are removed by aspiration followed by repeated washes. However, the shear force generated by such washing also detaches weakly adherent cells. In the minimal shear force adhesion assay (MSFA) described here, the removal of nonadherent cells is carried out by applying a gentle shear force in a fluid environment. In this procedure, adherent cells are not subjected to harsh and variable washing forces and are not exposed to surface tension caused by the removal of washing fluid between successive washes. Using the lymphoid cell lines XC1.5/51 and MPC11, the number of adherent cells determined by this new adhesion assay is three times higher than the conventional adhesion assay. This MSFA assay is simple, consistent, and easy to perform. With modifications for applying a defined shear force, this assay can be adopted to compare cell adhesion strength to various substrata.

Inhibition of platelet aggregation by diterpene acids from Pinus massoniana resin.

The acidic fraction of the resin of Pinus massoniana Lamb. from China was converted to the p-nitrophenyl esters, and the esters separated by chromatography. The separated p-nitrophenyl esters were individually hydrolysed by potassium hydroxide in acetone-water at room temperature to 8 diterpene acids of the pimarane and abietane groups: pimaric acid (8(14),15-pimaradien-18-oic acid) (1), levopimaric acid (8(14),12-abietadien-18-oic acid) (2), palustric acid (8,13-abietadien-18-oic acid) (3), neobietic acid (8(14),13(15)-abietadien-18-oic acid) (4), abietic acid (7,13-abietadien-18-oic acid) (5), dehydroabietic acid (8,11,13-abietatrien-18-oic acid) (6), 7-oxodehydroabietic acid (7-oxo-8,11,13-abietatrien-18-oic acid) (7) and 7 alpha-hydroxydehydroabietic acid (7 alpha-hydroxy-8,11,13-abietatrien-18-oic acid) (8). The structure (and stereochemistry) of the diterpene acids were substantiated by nuclear magnetic resonance spectroscopy (proton and carbon-13, one and two dimensional), by mass spectrometry (electron impact and methane chemical ionization) and by rotation measurements. The 8 diterpene acids were tested for their ability to inhibit the aggregation of washed rabbit platelets induced by platelet activating factor (PAF), adenosine diphosphate (ADP) and by calcium ionophore A23187. With platelet aggregation induced by the latter two agonists, activities comparable with or higher than linolenic acid were given by the first 4 acids. With aggregation induced by PAF, the first 3 acids show activity, but at a level significantly lower than that of linolenic acid. Levopimaric acid has the highest activity among the diterpene acids tested. It is proposed that this activity is related to the folded shape of the molecule.

13C NMR determination of the tautomeric and ionization states of folate in its complexes with Lactobacillus casei dihydrofolate reductase.

13C NMR studies provide a convenient way of obtaining detailed information about tautomeric and ionization states in protein-ligand complexes provided that suitably 13C-labeled molecules are available. In the present study, [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid were synthesized and the 13C NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analyzed as a function of pH. From these data it was possible to determine the tautomeric and ionization states of the bound folate and to obtain further evidence about the orientation of the pteridine ring in the complexes. In the 13C spectra of the ternary complexes of the 13C-labeled folic acids with DHFR and NADP+, each labeled carbon gave rise to multiple signals, confirming our previous findings that there are three interconverting conformational forms of bound folate (forms I, IIa, and IIb) in the ternary complex (Birdsall et al., 1989b). The 13C spectra of the binary complexes of folate and DHFR also provide direct evidence for the presence of forms IIa and IIb and indirect evidence of some form I at low pH values ( < 5.0). 2D 1H-13C HMQC-NOESY experiments on ternary complexes formed using the [2,4a,7,9-13C]folic acid were used to obtain intermolecular NOEs between the folate H7 proton and protons on the protein, and these provided further characterization of the orientations of the pteridine ring in the different bound forms of folate (form IIb with its pteridine ring in the catalytically active conformation and forms I and IIa with their pteridine rings turned over by 180 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid.

13C NMR studies of 13C-labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9-13C]- and [2,4a,6-13C]methotrexate (MTX) and [4,6,8a-13C]- and [2,4a,7,9-13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180 degrees difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4-NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR-MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.

Basement membrane and its components on lymphocyte adhesion, migration, and proliferation.

During inflammation and recirculation, lymphocytes migrate into tissues by traversing the capillary endothelium, a process known as extravasation. After crossing the endothelial cells, lymphocytes come into contact with the basement membrane, which is a specialized layer of extracellular matrix containing predominantly laminin, collagen type IV, entactin, and heparan sulfate proteoglycans. In tissue invasion by inflammatory cells and metastatic tumor cells, the basement membrane serves as a substratum for cell adhesion and migration. However, the role of basement membrane in lymphocyte extravasation remains unclear. In this study, we investigated the effect of basement membrane on lymphocyte adhesion, migration, and proliferation, using matrigel as a model for basement membrane. We observed that matrigel promotes both lymphocyte adhesion and migration, with entactin primarily responsible for promoting adhesion and laminin for promoting migration. In addition, activation of lymphocytes by anti-CD3 enhances their adhesion and migration on matrigel-coated substratum. We also observed that matrigel inhibits the proliferation of lymphocytes stimulated by Con A. Furthermore, we demonstrated that laminin is the matrigel component responsible for inhibiting lymphocyte proliferation. However, matrigel has no effect on the proliferation of lymphocytes stimulated by LPS. These results suggest that matrigel has different effects on lymphocyte subpopulations. In agreement with the results on proliferation, matrigel also inhibits the production of IL-2 by Con A-stimulated lymphocytes.

Syntactic complexity and adults' running memory span.

This study investigated age group differences in adults' running memory span for prose. College students and adults 60 to 94 years of age listened to a prose passage that was interrupted occasionally by pauses. At each pause, the adults attempted to recall the immediately preceding text. The pauses followed either two single-clause sentences, a two-clause right-branching sentence, or a two-clause left-branching sentence. There was a significant Age Group x Syntactic Form x Clause Order interaction such that the age group differences in verbatim recall were exacerbated by the effects of syntactic complexity. The elderly recalled 25% fewer words from the first embedded clause of the left-branching sentences than the college students, whereas they recalled only 4% fewer words from the first of two successive single-clause sentences. Performance on the running memory span task was also correlated with two measures of the adults' working memory: forward digit span and backward digit span. The pattern of correlations indicated that working memory limitations determine adults' running memory span for prose and contribute to age-group deficits in comprehension.

The expression of granulocyte/macrophage colony-stimulating factor in activated mouse lymphocytes declines with age.

The expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) was studied in spleen lymphocytes isolated from male C57BL/6J mice of 6, 20, and 29 months of age. GM-CSF expression (biological activity and mRNA level) was maximum after culturing the lymphocytes for 45 hr with concanavalin A and phorbol myristate acetate. The induction of both GM-CSF activity and mRNA levels was observed to decline over 60% between 6 and 29 months of age. The age-related decline in the level of GM-CSF paralleled the age-related decline in the mRNA levels of interleukin-2 and interleukin-3.

Haptotactic activity of fibronectin on lymphocyte migration in vitro.

In addition to mediating cell adhesion, fibronectin (FN) also affects the migration of different cell types. However, the role of FN in lymphocyte migration is unclear. In this study, we examined the effects of FN on the in vitro migration of lymphocytes. Using the checkerboard analysis in a blind-well microchemotaxis assay, soluble FN was determined to have neither a chemotactic nor chemokinetic effect on spleen or thymus lymphocytes. However, when the nitrocellulose filter was coated unidirectionally with FN, the migration of both spleen and thymus lymphocytes into the filter was enhanced, indicating that FN is haptotactic for lymphocytes. When the filter was coated bidirectionally, no enhancement in migration was observed, indicating that FN is not haptokinetic for lymphocytes. When the FN cell-binding domain and the heparin-binding domain were tested, the cell-binding domain was haptotactic for both spleen and thymus lymphocytes, whereas the heparin-binding domain was only haptotactic for spleen lymphocytes. Because the heparin-binding domain can mediate strong adhesion of thymus lymphocytes, the lack of haptotactic activity is likely to be the result of excessive binding that prevents cell motility.

A urinary cysteine-halothane metabolite: validation and measurement in children.

An attempt was made in children to identify a urinary halothane-cysteine conjugate which had been described previously in adult patients following administration of halothane. If this conjugate was found it would indicate that a reductive metabolite of halothane binds covalently with the sulphydryl-containing amino acid, cysteine, a reaction which could lead to hepatic injury. The potential halothane-cysteine conjugate, N-acetyl-S-(2-bromo-2-chloro-1,1-difluoroethyl)-L-cysteine (acetyl BCFEC), was prepared and the identity of the compound established using hydrogen-1 and carbon-13 NMR spectroscopy and methane chemical ionization mass spectrometry. A measurement technique for acetyl BCFEC was developed using HPLC with u.v. detection at 200 nm. In six children after halothane anaesthesia, one child being studied twice, urine was collected for up to 1 week and analysed for acetyl BCFEC. Little or no acetyl BCFEC was detected in any of the 43 urine samples tested, indicating that in children it is not a significant urinary metabolite of halothane.