Polyglutamate derivatives of folic acid coenzymes and methotrexate.
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Animals↗
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Effect of plasma glow, glutaraldehyde and carbodiimide treatments on the enzymic degradation of poly (L-lactic acid) and poly (gamma-benzyl-L-glutamate) films.
The hydrolytic and enzymic degradation of poly(L-lactic acid) (PLA) and poly(gamma-benzyl L-glutamate) (PBGA) films, together with a series of surface treatments, were studied, as a function of exposure time. The degradation of these polymers was monitored by weight loss, contact angle, pH changes and tensile strength studies. Glutaraldehyde treatment retained the maximum strength of PLA in buffer, followed by carbodiimide, compared with control films. On the other hand, plasma glow reversed the effect. The ability of alpha-chymotrypsin, carboxypeptidase, ficin, esterase, bromelain and leucine aminopeptidase to modulate the degradation of PLA and PBGA was also investigated. Addition of these enzymes to the polymer-buffer system reduced the tensile strength of these polymers variably. Among the six enzymes studied, leucine aminopeptidase showed the highest enzymic effect on the degradation of the glutaraldehyde-treated and bare PLA or bare PBGA films. However, glutaraldehyde-cross-linked PLA demonstrated maximum stability in buffers or in all other enzyme systems studied compared with bare PLA. It is conceivable that surface treatments on these polymers might have altered their physical and chemical configuration and the subsequent degradation properties. Surface modifications may provide new ways of controlling the biodegradation of polymers for a variety of biomedical applications.
Biocompatible Materials↗
Targeted taxane therapy for cancer.
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Antineoplastic Agents↗
Salt-dependent conformational diversity of alternating poly (Glu-Leu).
Alternating poly(Glu-Leu) was synthesized by the condensation of the corresponding dipeptide p-nitrophenyl ester at high concentration. It exhibits a random coil structure in pure water at neutral pH. Addition of monovalent cations, such as NH4+ to a final 0.1 M solution, induces a transition to a water soluble beta-structure. The salt effect is quite selective since no transition was observed with Li+, Na+ or Cs+ ions. Addition of 0.5 equiv of calcium, cobalt or manganese chlorides per glutamyl residue induces similar coil to beta-sheet transitions. No polymer precipitation was observed at these very low salt concentrations. Addition of 0.5 equiv. of Cu2+ or 0.15 equiv. of Fe3- induces a coil to alpha-helix transition. Molecular modeling has been used to understand tentatively the main factors controlling the different conformations observed with the various metal ions.
Ammonia↗
Interaction of polyglutamyl derivatives of methotrexate, 10-deazaaminopterin, and dihydrofolate with dihydrofolate reductase.
Polyglutamyl derivatives of methotrexate (MTX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef liver. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MTX causes stepwise decreases in the concentration required for 50% inhibition (IC50) with each additional Glu residue until MTX with a total of six Glu residues has an IC50 value 1/3 that of MTX. With 10-DAM the pattern is more complex. The IC50 values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five Glu residues have progressively lower IC50 values, the latter being equipotent with 10-DAM. With dihydropteroylpentaglutamate as substrate instead of dihydrofolate, the IC50 values are increased 2- to 5-fold for both MTX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KCl to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.
Aminopterin↗
Determinants of resistance to antifolates: biochemical phenotypes, their frequency of occurrence and circumvention.
Biochemical alterations associated with acquired resistance of tumor cells to antifolates are diverse and multiple in number. These most often have included both quantitative and qualitative alterations at the level of membrane transport and of the primary intracellular target, dihydrofolate reductase (DHFR). More recent studies suggest determining biochemical alterations at the level of thymidylate synthase activity and 4-aminofolate polyglutamylation. Approaches to the circumvention of acquired antifolate resistance at the level of new drug design are described which incorporate a kinetic analysis of the various biochemical phenotypes and a systematic analysis of their structure-activity relationships. A consideration of the relative frequency of occurrence of individual phenotypes during therapy is also included. This introduces the notion of population genetics in an evaluation of resistance phenomenon and of clinically significant approaches for its circumvention.
Biological Transport↗
Vibrational analysis of peptides, polypeptides, and proteins. XXXII. alpha-Poly(L-glutamic acid).
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Peptides↗
Diagnostic and therapeutic potential of poly(benzyl L-glutamate).
Poly(benzyl L-glutamate) (PBLG) microcapsules, prepared by a solvent evaporation technique for intravenous injection, are evaluated for their potential use in diagnostic computed tomographic enhancement of liver images. The smaller microcapsules, < 3 microns, loaded with a radiopaque contrast material, ethyl iopanoate (IOPAE), produced prolonged opacification of the liver when delivered intravenously. In vivo tissue distribution studies of PBLG-131I-IOPAE (5 microCi/rat, iv) showed that liver had the highest uptake (percent of injected dose/g of tissue) among other organs 24 h postinjection. An in vitro estrogen receptor assay in pig uteri indicated that PBLG conjugated with estrone did not interfere with estrogen receptor affinity, suggesting the estrogen therapy potential of PBLG-estrone.
Animals↗
Synthesis of AB diblock copolymers by atom-transfer radical polymerization (ATRP) and living polymerization of alpha-amino acid-N-carboxyanhydrides.
The synthesis of poly(methyl acrylate)-block-poly(gamma-benzyl-L-glutamate) (PMA-b-PBLG) diblock copolymers, using atom-transfer radical polymerization (ATRP) of methyl acrylate and living polymerization of gamma-benzyl-L-glutamate-N-carboxyanhydride (Glu-NCA) is described. Amido-amidate nickelacycle end groups were incorporated onto amino-terminated poly(methyl acrylates), and the resulting complexes were successfully used as macroinitiators for the growth of polypeptide segments. This method allows the controlled preparation of polypeptide-block-poly(methyl acrylate) diblock architectures with control over polypeptide chain length and without the formation of homopolypeptide contaminants.
Amino Acids↗
Biochemistry and molecular genetics of poly-gamma-glutamate synthesis.
Current research into poly-gamma-glutamate (PGA) and its biosynthesis is reviewed. In PGA-producing Bacillus subtilis, glutamate racemase supplies abundant DL-glutamate, the substrate for PGA synthesis. The pgsBCA genes of PGA-producing B. subtilis, which encode the membrane-associated PGA synthetase complex PgsBCA, were characterized and the enzyme complex was suggested to be an atypical amide ligase based on its structure and function. A novel reaction mechanism of PGA synthesis is proposed.
Amino Acid Isomerases↗
Metabolism of the diaminoantifolates: biosynthesis and pharmacology of the 7-hydroxyl and polyglutamyl metabolites of methotrexate and related antifolates.
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Aminopterin↗
Characterization of polyamino acids by use of GPC-viscometry technology.
The properties of polybenzyl glutamate, a well known polyamino acid vary considerably with molecular weight in both solid state and in solution. Therefore, accurate determinations of the molecular weights of these polyamino acids is essential. The dual viscometer/refractometer when used as detector system for size exclusion chromatography provides a way of determining accurate molecular weights. An indirect method of determining the molecular weight distribution (MWD) and the radius of gyration distribution (RgD) of polybenzyl glutamate is described. The MWD is calculated from the measured value of intrinsic viscosity (IV) and the known IV-to-MW relationship, at every SEC retention volume slice. Such a technique of determining MWD requires no calibration and is more precisely measurable than conventional SEC methods.
Amino Acids↗
Glr, a glutamate racemase, supplies D-glutamate to both peptidoglycan synthesis and poly-gamma-glutamate production in gamma-PGA-producing Bacillus subtilis.
Poly-gamma-glutamate (gamma-PGA)-producing Bacillus subtilis contains two glutamate racemase genes, glr and yrpC, as does gamma-PGA-nonproducing B. subtilis strain 168. glr and yrpC on the chromosome of gamma-PGA-producing strain r22 were separately disrupted by means of gene replacement with an erythromycin resistance determinant. yrpC-disruption caused no effects on growth or gamma-PGA-production, whereas glr was disrupted only when an exogenous glr copy was present on a plasmid. In addition, the D-glutamate content of gamma-PGA produced by the yrpC-disruptant was the same as that produced by the parental strain r22. Glr in strain r22 is therefore responsible for the supply of D-glutamate to the synthesis of both peptidoglycan and gamma-PGA. Consistent with this idea, glr was transcribed actively during the exponential growth phase for peptidoglycan synthesis and continuously at a low, but distinct, level during the stationary phase for gamma-PGA production, whereas yrpC was transcribed at a very low level throughout growth. Phylogenetic analysis of glutamate racemases from eubacteria showed that YrpC is distinct from other glutamate racemases.
Amino Acid Isomerases↗
Transdermal iontophoresis: modulation of electroosmosis by polypeptide.
The objective of this research was to further evaluate the relative importance of electrorepulsion and electroosmosis to the mechanism of enhanced transport across the skin during iontophoresis. Specifically, the impact of iontophoresing into the skin positively and negatively charged polypeptides (poly-L-lysines and poly-L-glutamic acids, respectively) on the membrane's permselectivity and hence on the quantity and direction of electroosmotic flow, was examined. Experiments were performed in vitro at pH 7.4 using conventional methodology; electroosmosis during the iontophoresis of the polypeptides into and across the skin was tracked in the usual way via the movement of the polar, uncharged, non-metabolizable marker, D-mannitol. Electrotransport of the cationic polypeptides attenuated electroosmotic flow in the normal anode-to-cathode direction; the degree of inhibition was correlated both with the initial concentration of poly-L-lysine in the anodal chamber and with the molecular weight of the polypeptide employed (from 1 to 25 kilodaltons). Iontophoresis of the anionic poly-L-glutamic acids from the cathode provoked a slight increase in electroosmotic flow in the 'reverse' direction (i.e. from the receptor phase beneath the skin towards the cathode chamber located on the epidermal side of the membrane); this effect, however, was much less dramatic than that produced in the opposite sense by the cationic polypeptides. The results suggest that driving large positively-charged polypeptide molecules into the skin leads to neutralization of the membrane's negativity, a subsequent loss of permselectivity and a concomitant attenuation of electroosmosis in the conventional anode-to-cathode direction. Presumably, the relatively poor iontophoretic permeability of these species (which becomes more and more evident with increasing molecular weight) results in a sufficiently important association of the polypeptide with the skin during the period of current passage. Much less significant effects are realized by the cathodal iontophoresis of poly-anions due to the difficulty of 'pushing' negative ions into an already negatively-charged membrane.
Administration, Cutaneous↗
Glutamate receptors are expressed by bone cells and are involved in bone resorption.
The excitatory amino acid glutamate (Glu) is a potent neurotransmitter in the central nervous system and exerts its action via a variety of glutamate receptors (GluRs). Because we had previously shown that a poly-glutamate (poly-Glu) peptide stimulates bone resorption in vitro, an effect specific to Glu (Raynal, C., Delmas, P. D., and Chenu, C. Bone sialoprotein stimulates in vitro bone resorption. Endocrinology 137:2347-2354; 1996), we investigated the possibility that bone cells express GluRs, and whether they may be important for osteoclast activity. Using immunocytochemistry on rat bone sections, we have shown that all mature bone cells (osteoblasts and osteoclasts) express GluRs, and that the ionotropic N-methyl-D-aspartate (NMDA) receptor 1 subunit (NMDAR1) appears most highly expressed. Osteoclasts isolated from rabbit long bones also possessed NMDAR1 GluRs. Bone resorption in vitro by isolated osteoclasts was inhibited by a monoclonal antibody directed against NMDAR1, as well as by two pharmacological antagonists of this receptor (D-AP5 and MK 801), which also antagonized poly-Glu stimulated bone resorption. These results suggest a possible new mechanism for regulating osteoclast activity and indicate that excitatory amino acids such as glutamate may be important local regulators of bone cell functions.
2-Amino-5-phosphonovalerate↗
Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism.
We have solved crystal structures of two complexes with Escherichia coli thymidylate synthase (TS) bound either to the cofactor analog N10-propargyl-5,8-dideazafolate (CB3717) or to a tighter binding polygutamyl derivative of CB3717. These structures suggest that cofactor binding alone is sufficient to induce the conformational change in TS; dUMP binding is not required. Because polyglutamyl folates are the primary cofactor form in vivo, and because they can bind more tightly than dUMP to TS, these structures may represent a key intermediate along the TS reaction pathway. These structures further suggest that the dUMP binding site is accessible in the TS-cofactor analog binary complexes. Conformational flexibility of the binary complex may permit dUMP to enter the active site of TS while the cofactor is bound. Alternatively, dUMP may enter the active site from the opposite side that the cofactor appears to enter; that is, through a portal flanked by arginines that also coordinate the phosphate group in the active site. Entry of dUMP through this portal may allow dUMP to bind to a TS-cofactor binary complex in which the complex has completed its conformational transition to the catalytically competent structure.
Binding Sites↗
Photolithographic patterning of dendrimer monolayers and pattern-selective adsorption of linear macromolecules.
Alkyl groups of n-octadecyltrimethoxysilane (ODS) in a self-assembled monolayer on a silicon substrate were oxidized to carboxyl groups by partial irradiation of vacuum ultra-violet light under the photomask, producing a COOH/ODS line pattern. After active esterification of carboxyl groups, two kinds of amine-terminated dendrimers, poly(propyleneimine) and poly(amido amine) (PAMAM) dendrimers, were immobilized on a COOH line through amide-bond so that photolithographic dendrimer/ODS pattern was finally fabricated. Preparation was certified by atomic force microscopy (AFM) and surface-enhanced infrared absorption spectroscopy at transmission mode. Adsorption of linear macromolecules was examined on PAMAM dendrimer/ODS pattern. After adsorption of poly-L-glutamic acid (PGA) at a pH below alpha-helix--random coil transition, rod-shape texture was observed only on the dendrimer line in an AFM image. This texture is an aggregate of alpha-helical PGA. Sodium hyaluronate and DNA were also adsorbed selectively on the dendrimer line, keeping the line profile, although characteristic textures were not observed.
Adsorption↗
Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons.
Polyglutamylation is an important posttranslational modification of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin heterogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated alpha- and beta-tubulin occurring during neuronal differentiation in culture. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation and contributes to increase the complexity of tubulin in mature neurons.
Animals↗