Biomedical subjects
B Lippert
Publications and source records attributed to B Lippert.
Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.
Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.
Trans-diammineplatinum(II): what makes it different from cis-DDP? Coordination chemistry of a neglected relative of cisplatin and its interaction with nucleic acids.
The question raised, i.e., "trans-diammineplatinum(II)--what makes it different from its cis isomer?" permits a number of answers, relating to various aspects of the chemistry of the two compounds, but the crucial one concerning antitumor activity is yet to be answered. In principle, any of the following reasons or combinations thereof might account for the observed differences in biological effects, but some are more likely than others: 1. TARGET MOLECULES: It is generally accepted that DNA is the most important target molecule of cisplatin and that inhibition of DNA synthesis correlates with antitumor activity. This fact does not contradict observations on extensive reaction with other biomolecules. The mutagenic effects of trans-a2Pt(II), although weaker than for cis-DDP, point toward DNA as being an important target for trans-DDP as well. 2. DNA ADDUCTS: As a consequence of the inherent difference in geometry, the two isomers form different adducts. Specifically, trans-DDP cannot form 1,2-intrastrand crosslinks, which represent by far the most abundant adducts of cis-DDP. On the other hand, the trans isomer displays a greater variation in nucleobase donor sites (cf. Sec. 4.2). 3. DNA STABILITY: While the 1,2-intrastrand adducts of cis-DDP, regardless if GG or AG, consistently cause DNA kinking and lead to thermal destabilization, the effects of trans-DDP adducts seem to be of a greater variability, causing thermal stabilization or destabilization. For intrastrand 1,3 adducts in puXpu sequences, the intervening base X appears to be important in this respect (cf. Sec. 4.3). 4. DNA REPAIR: In living cells, higher doses of trans-DDP as compared to cis-DDP are required to bind an equal number of Pt atoms per nucleotide [66]. On the other hand, bifunctional DNA adducts of either isomer inhibit DNA replication to the same extent [66,150,151]. This finding has been interpreted in terms of a differential repair of adducts of the two isomers [66], but an alternative explanation has also been offered [152]. It is to be noted that repair of monofunctional trans-DDP lesions does not require enzymatic repair but rather may be accomplished by any nucleophile within the cell exercising a reasonably high trans influence, e.g., an S-donor of glutathione. 5. INTRINSIC REACTIVITY: Differences in hydrolysis kinetics of the two isomers (unfavorable equilibrium concentration of trans-[(NH3)2Pt-Cl(OH2)]+ [37]) and in reaction rates of various hydrolysis species with DNA constituents [36] could, in principle, explain a difference in biological effects. On the other hand, the kinetics of reactions of the dichloro species of both isomers with DNA (in the absence of any repair agents) appear not to be that largely different to produce a strong point for differential reactivity of this species. Model studies have shown at least one more distinct difference between mono(nucleobase) adducts of both isomers: While cis-[a2PtLCl]+ can lose the amine trans to Cl, in trans-[a2PtLCl]+ Cl is capable of displaying L (cf. Sec. 6.1). Both reactions are not very fast with Cl-, but any good nucleophile replacing Cl- would do so with high efficiency. In the case of trans-DDP, such a reaction leads to removal of Pt from DNA, unlike in the case of cis-DDP (even though we are aware that cis effects may also be operative). With respect to amine displacement from monoadducts of cis-DDP, it is interesting to speculate on the fate of (toxic) NH3. Is it capable of undergoing condensation reactions with biomolecules? In theory, such a scenario might provide possibilities for the role of the amine ligands in cis-(amine)2Pt(II) compounds alternative to those commonly accepted (e.g., role of NH protons in stabilizing DNA adducts [153]). 6. TRANSPORT AND DISTRIBUTION: Despite differences in water solubility of the two isomers (trans-DDP less soluble), this property is unlikely to be important at physiological concentrations...
Metal ions in multiple-stranded DNA.
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[Objective damage and subjective discomfort after general anesthesia--a comparison of intubation and laryngeal mask].
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From cisplatin to artificial nucleases--the role of metal ion-nucleic acid interactions in biology.
Metal ions and metal coordination compounds bind to nucleic acids in a variety of ways, ranging from weak electrostatic interactions via hydrogen bonding and/or van der Waals forces to strong covalent binding. Metal ions naturally take part in the formation and the degradation of nucleic acids, and the propensity of certain metal coordination compounds to bind to nucleic acids, notably DNA, is exploited in cancer chemotherapy. Moreover, metal compounds have a wide potential as chemical probes for nucleic acid structures and as tools for nucleic acid processing.
2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase.
It has been found that 2'-deoxy-2'-methyleneuridine (MdUrd), 2'-deoxy-2'-methylenecytidine (MdCyd), and 2'-deoxy-2',2'-difluorocytidine (dFdCyd) 5'-diphosphates (MdUDP (1) MdCDP (2) and dFdCDP (3), respectively) function as irreversible inactivators of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). 2 is a much more potent inhibitor than its uridine analogue 1. It is proposed that 2 undergoes abstraction of H3' to give an allylic radical that captures a hydrogen atom and decomposes to an active alkylating furanone species. RDPR also accepts 3 as an alternative substrate analogue and presumably executes an initial abstraction of H3' to initiate formation of a suicide species. Both 2 and 3 give inactivation results that differ from those of previously studied inhibitors. The potent anticancer activities of MdCyd and dFdCyd indicate a significant chemotherapeutic potential. The analogous RDPR of mammalian cells should be regarded as a likely target and/or activating enzyme for these novel mechanism-based inactivators.
Lactate dehydrogenase isoenzymes in squamous cell carcinomas of the oral cavity.
Lactate dehydrogenase (LDH; EC 1.1.1.27) isoenzymes in human epithelial cells from squamous cell carcinomas and healthy tissues of the oral cavity of five patients were analyzed using isoelectric focussing. A new basic isoenzyme and high LDH-7 and LDH-9 activity were found in tumor cells in contrast to epithelial cells of adjoining nontumor tissue. These findings indicate that gradual changes in the percentage distribution of LDH isoenzymes may represent a useful parameter of disease activity in patients with squamous cell carcinomas.
Inhibited methionine incorporation in human squamous carcinomas of the oral cavity as a measure for response to 5-fluorouracil.
Human epithelial cells and fibroblasts from malignant tumors and healthy tissues of the oral cavity were exposed in vitro to increasing concentrations of 5-fluorouracil. In order to determine whether the two cell types showed a difference in uptake, accumulation was measured over time using tritiated fluorouracil. Maximal drug uptake occurred in highly proliferative epithelial cells from cancerous tissue at 3 h, whereas fibroblasts from the same tissue and other cell types from healthy tissue exhibited a steadily increasing uptake over a long period of time. By use of [3H]methionine, a direct correlation could be demonstrated between the amount of incorporated drug and the fall in the rate of de novo protein synthesis. Furthermore, suppression of protein synthesis was closely related to the inhibition of cell division. These results indicate that the level of de novo protein synthesis can be a direct measure of the anticancer effect of fluorouracil.
Effect of dextran-bound acetazolamide on rabbit corneal endothelial ion fluxes.
The effects of a carbonic anhydrase inhibitor, acetazolamide, bound to a 72,000 dalton dextran (DBI) on bicarbonate and sodium fluxes across the isolated rabbit corneal endothelium have been examined. When DBI was present on the aqueous-facing endothelial surface, there was a marked inhibition of both the stromal to endothelial unidirectional and net flux of bicarbonate. This suggests that the exit of bicarbonate from the cell into the aqueous-facing solution is influenced by the enzyme carbonic anhydrase. No change was found in sodium fluxes under these same incubation conditions. When DBI was present on the stromal-facing surface of the endothelium, no changes were found in unidirectional or net bicarbonate fluxes; the sodium flux from stroma to endothelium was increased, however, with no change in net flux. This data implies that the link between sodium and bicarbonate movement across the endothelium is not a direct coupling between the transport of the two ions in the form of a symport (Na+:HCO3-) at the apical cell border.
Enzyme-activated irreversible inhibition of rat and mouse brain 4-aminobutyric acid-alpha-ketoglutarate transaminase by 5-fluoro-4-oxo-pentanoic acid.
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Stereochemistry of reactions catalysed by mammalian-brain L-glutamate 1-carboxy-lyase and 4-aminobutyrate: 2-oxoglutarate aminotransferase.
Deamination of 4-aminobutyrate by mammalian or bacterial 4-aminobutyrate aminotransferases involves the abstraction of the pro-S hydrogen on C-4 of 4-aminobutyrate. Decarboxylation of L-glutamate by rat brain glutamate decarboxylase occurs with retention of configuration. Inhibition of this enzyme by (S)-4-aminohex-5-ynoic acid involves the abstraction of the proton at C-4 of the inhibitor. On the basis of this finding, we postulate the existence of an abnormal reaction of glutamate decarboxylase in which the proton at C-4 of (S)-4-aminohex-5-ynoic acid is removed in a manner similar to the one which normally occurs in enzymatic transaminations of L-amino acids. This reaction is presumably facilitated by the acetylenic group adjacent to the eliminated proton.
Selective irreversible inhibition of mammalian histidine decarboxylase by alpha-chloromethyl histidine [proceedings].
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The effect of gamma-acetylenic GABA, an enzyme-activated irreversible inhibitor of GABA-transaminase, on dopamine pathways of the extrapyramidal and limbic systems.
gamma-Acetylenic GABA (100 mg/kg i.p.) inhibited GABA-transaminase activity and caused a several-fold increase in the concentration of GABA in rat brain. This increased GABA concentration was associated with a decreased rate of dopamine depletion following alpha-methyl-p-tyrosine treatment and a decrease in homovanillic acid in extrapyramidal and limbic structures suggesting a decrease in dopamine turnover in both pathways. In addition, gamma-acetylenic GABA injected into the ventral mesencephalic tegmentum decreased dopamine turnover in the mesolimbic forebrain. These results are consistent with a modulatory function of GABAergic neurons on extrapyramidal and limbic dopamine pathways. Inhibitory effects on dopaminergic functions of the extrapyramidal and limbic systems were also indicated by the amphetamine and apomorphine-induced ipsilateral turning after unilateral substantia nigral injections of gamma-acetylenic GABA and by the attenuation of dopamine-induced hypermotility after bilateral injections of gamma-acetylenic GABA into the nucleus accumbens.
Mechanism of the stereospectific irreversible inhibition of bacterial glutamic acid decarboxylase by (R)-(--)-4-aminohex-5-ynoic acid, an analogue of 4-aminobutyric acid.
4-Aminohex-5-ynoic acid inhibits bacterial glutamic acid decarboxylase in a time-dependent irreversible manner. The inhibition is stereospecific and requires the abstraction of the propargylic hydrogen from 4(R)-(--)-4-aminohex-5-ynoic acid. This leads to the generation of a reactive alkylating agent in the active site which can react with a nucleophilic residue. At complete inhibition, there is incorporation of one molecule of inhibitor per pyridoxal binding site. If the decarboxylation of glutamate occurs with retention of configuration, the irreversible inhibition of this enzyme by the 4-(R) isomer can be rationalized on the basis of reversibility of the protonation step in the normal catalytic mechanism.
Molecular properties of the adrenergic alpha-receptor. I--Structural requirements for specific covalent occupancy by N,N'-bis--(5-aminopentyl)cystamine derivatives.
The synthesis and alpha-adrenoreceptor blocking activity of several substituted analogs of the prototype alpha-blocker N,N'-bis-(5-aminopentyl)cystamine (APC) are described. The three optical forms of the analog carrying methyl groups on the carbons alpha- to the sulfurs were synthesized and shown to be equipotent and somewhat less active than APC. The omega, omega'-bis-guanidino analog of APC was less active. Significant improvement in potency was observed only with APC analogs carrying benzyl and substituted benzyl groups on the terminal nitrogens. Linking the terminal nitrogens of APC with a p.xyledenyl group so as to give the 26-membered analog caused a sharp drop in activity. The significance of these results as regards the alpha-receptor topography is discussed.
4-amino-hex-5-enoic acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase in mammalian brain.
Incubation of rat brain 4-aminobutyrate aminotransferase with 4-amino-hex-5-enoic acid, a substrate analog of 4-aminobutyric acid, results in a time-dependent irreversible loss of enzymatic activity. In the presence of 0.1 mM inhibitor the half-life of the inactivation process is approximately 6 min. Low concentrations of L-glutamic acid or 4-aminobutyric acid protect against this inactivation, while 2-oxoglutarate prevents this protection, suggesting that only the pyridoxal form of the enzyme is susceptible to inhibition by 4-amino-hex-5-enoic acid. The irreversible inhibition of mammalian 4-aminobutyrate aminotransferase by 4-amino-hex-5-enoic acid is selective. There is no inhibition of this enzyme from Pseudomonas fluorescens with the inhibitor at mM concentrations. Even at 10 mM there is no irreversible inhibition of mammalian glutamate decarboxylase or of aspartate aminotransferase, while alanine aminotransferase is inhibited over 500 times more slowly than rat brain 4-aminobutyrate transaminase.
The effect of 4-amino hex-5-ynoic acid (gamma-acetylenic GABA, gammma-ethynyl GABA) a catalytic inhibitor of GABA transaminase, on brain GABA metabolism in vivo.
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