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A series of N-alkyl-N-methylpyrrolidinium halide salts have been synthesized and investigated as potentially useful and tunable detergents for a variety of applications.
Chemical analysis of a solid phase fermentation of an Australian Penicillium citrinum strain has returned all known examples of a rare class of N-methyl quinolone lactams, quinolactacins A2 (1), B2 (2), C2 (3) and A1 (4), together with the new quinolactacins B1 (5), C1 (6), D1 (7) and D2 (8), and the novel derivatives quinolonimide (9) and quinolonic acid (10). Complete stereostructures were assigned to all these compounds by detailed spectroscopic analysis and chemical interconversion. Carefully controlled and monitored decomposition studies have confirmed that quinolactacins readily undergo C-3 epimerization and oxidation, and under appropriate conditions convert to quinolonimide and quinolonic acid. Mechanisms for key transformations are proposed. The decomposition studies suggested that only quinolactacins A2 (1) and B2 (2) are genuine natural products, with all other isolated compounds being decomposition artefacts. Quinolactacins C1 (6), C2 (3), and the racemic mixture of quinolactacins D1/D2 (8/7) all displayed notable cytotoxic activity.
1. Peptide-elongation factors were purified from rat liver and treated with cholesterol esterase and phospholipase A2 immobilized on Sepharose 4B. 2. Binding of L-[3H]-phenylalanyl-tRNA to 40S ribosomal subunits was decreased by approx. 70% and to polyribosomes by 30% in the presence of the binding factor incubated with cholesterol esterase. Treatment of this factor with immobilized phospholipase A2 decreased the binding to smaller ribosomal subunits by only about 15%. 3. Poly(U)-dependent phenylalanine polymerization by ribosomal subunits was decreased to approx. 30% of its original value by treatment of both elongation factors with cholesterol esterase. 4. The normal activity of esterase-treated elongation factor in both the binding reaction and peptide-elongation assay was fully recovered by the addition of cholesteryl 14-methyl-hexadecanoate. 5. Different classes of lipids present in peptide-elongation factor 1 have apparently different functions. Whereas phospholipids are required to maintain the strcture of heavy aggregates of this factor, the presence of cholesteryl 14-methylhexadecanoate is obviously necessary for the normal function of peptide-elongation factors.
Molecular characterization of HLA class II expression was investigated in five lung tumour cell lines at the protein and mRNA levels. The cell lines exhibited a differential expression of HLA-DR, HLA-DP and HLA-DQ products and also showed differences in the inducibility of HLA class II genes by gamma-IFN. Gamma-IFN stimulation induced only HLA-DR expression to varying degrees in three cell lines, while only one cell line showed stimulation for HLA-DP and none for HLA-DQ antigens. These results suggest locus-specific regulation for the three loci. The presence of DR protein on the cell-surface membrane was always positively correlated with the presence of HLA-DR mRNA in the cells. After treatment with 5-azacytidine in the A549 cell line, which expressed the lowest values, there was no effect on HLA class II levels. This suggested that methylation does not play an important role in the lack of MHC class II antigen expression. In addition to studying mRNA levels of HLA class II antigens, we analysed mRNA of the proto-oncogene c-myc and observed a positive correlation of two mRNA: the increments in HLA-DR expression were associated with increments in c-myc expression. This suggests a relationship between the regulatory and HLA-DR antigens control the expression of c-myc and HLA-DR antigens in lung tumour cell lines.
Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O(6) position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O(6)-methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C.O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T.O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.
PF1022A belongs to a recently identified class of N-methylated cyclooctadepsipeptides (CODPs) with strong anthelmintic properties. Described here is the cell-free synthesis of this CODP and related structures, as well as the purification and enzymatic characterization of the responsible synthetase. For PF1022A synthesis extracts of Mycelia sterilia were incubated with the precursors L-leucine, D-lactate, D-phenyllactate, and S-adenosyl-L-methionine in the presence of ATP and MgCl(2). A 350-kDa depsipeptide synthetase, PFSYN, responsible for PF1022A synthesis was purified to electrophoretic homogeneity. Like other peptide synthetases, PFSYN follows a thiotemplate mechanism in which the substrates are activated as thioesters via adenylation. N-Methylation of the substrate L-leucine takes place after covalent binding prior to peptide bond formation. The enzyme is capable of synthesizing all known natural cyclooctadepsipeptides of the PF1022 type (A, B, C, and D) differing in the content of D-lactate and D-phenyllactate. In addition to PF1022 types A, B, C, and D, the in vitro incubations produced PF1022F (a CODP consisting of D-lactate and N-methyl-L-leucine), as well as di-, tetra-, and hexa-PF1022 homologs. PFSYN strongly resembles the well documented enniatin synthetase in size and mechanism. Our results suggest that PFSYN, like enniatin synthetase, is an enzyme with two peptide synthetase domains and forms CODP by repeated condensation of dipeptidol building blocks. Due to the low specificity of the d-hydroxy acid binding site, D-lactate or D-phenyllactate can be incorporated into the dipeptidols depending on the concentration of these substrates in the reaction mixture.
During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.
Mutational spectra induced by different heterocyclic amines were characterized and compared with those obtained from diethylnitrosamine and N-methyl-N-nitrosourea. Mutation classes were identified by means of a series of mutant lacZ genes in F' episomes in Escherichia coli engineered to detect specifically each of two transitions, four transversions and five kinds of frameshift events. More than 99.5% of the mutations induced by heterocyclic amines were frameshift mutations. -2(C.G-G.C) frameshifts were favored over other types, such as +1(G.C), -1(G.C), +1(A.T) and -1(A.T), except when 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was administered. -1(G.C) and +1(G.C) frameshifts predominate following Trp-P-1 treatment. A small number of G.C-->T.A transversions were induced by the treatment with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline as well as with several other heterocyclic amines examined. Since G.C-->T.A transversions, but not frameshift mutations, are reported to play a role in heterocyclic amine-induced activation of the c-Ha-ras protooncogene or inactivation of the p53 tumor suppressor gene, the low level of base substitutions, particularly G.C-->T.A transversions, may represent a partial explanation for the relatively modest carcinogenic activity of heterocyclic amines, despite their extraordinarily strong mutagenicity in the Salmonella mutation assay.
The region surrounding a focal cerebral infarct shows selective rather than generalized neuronal damage. The "ischemic penumbra" has been defined electrophysiologically as that region which shows a loss of electrical activity while retaining some metabolic viability (Astrup et al., Stroke, 1981:6:723-5). How factors that contribute to ischemic damage may cause such selective damage to subpopulations of neurones will be discussed. Particular attention will be directed to the cortical pyramidal cells, thought to be a major component of cerebral electrical activity. The N-methyl-D-aspartate (NMDA) class of glutamic acid receptors appears to have a role in certain aspects of ischemic damage, mediated through excitotoxins and calcium ions. The regulation of this receptor complex appears to be unaffected by ischemia, and thus it may provide a target for possible therapeutic interventions. Selective neuronal loss tends to be associated with neurotransmitter imbalance; therefore, treatments designed to correct this imbalance by affecting the activity of those cells less sensitive to ischemia may provide an alternative approach to establish normal cerebral function.
The survival of cerebellar granule cells in culture is stimulated by activation of the N-methyl-d-aspartate (NMDA) class of glutamate receptors. Activation of these receptors at the key period for cell survival in vitro (3 days; 3DIV) resulted in a sustained elevation of intracellular free calcium concentration [Ca2+]i over the same concentration range of NMDA that led to granule cell survival. Agents that release Ca2+ from intracellular stores led to only small, transient elevations of [Ca2+]i and were unable to stimulate granule cell survival. Addition of the Ca2+ ionophore ionomycin to granule cell cultures at 3DIV resulted in increased granule cell number at 7DIV. The ability of ionomycin to stimulate granule cell survival was related to the [Ca2+]i elicited, indicating that a rise in [Ca2+]i is sufficient to activate the processes leading to granule cell survival and that the extent of the elevation in [Ca2+]i is crucially important in determining granule cell fate.
We have analysed the factors which regulate MHC class II expression in mouse T cell lines. Two such lines, BW 5147 and PLT-24.2, were used in this study. Using 5-azacytidine (5 AzaC) we have shown that hypomethylation of DNA can induce class II antigen synthesis in BW 5147. The expression of class II in PLT-24.2 cells seems to be under a different control mechanism. Southern blot analysis of I-A beta gene in PLT-24.2 suggests that the expression of class II in this cell line is probably the outcome of a gene rearrangement. We hypothesise that insertion of viral long terminal repeats (LTR) next to the class II genes in transformed T cell lines can act as a promoter for the expression of class II antigens.
Although there are many analgesics on the market for the treatment of nociceptive pain, there are none with FDA approval for the treatment of neuropathic pain. With a better understanding of the anatomy and physiology of pain, there is a significant effort in developing new drugs that interact specifically with pain pathways. This higher drug specificity is likely to result in drugs that are more efficacious with fewer side effects. This has led to the development of many drugs for the treatment of neuropathic pain. These drugs are divided into the following therapeutic classes: 1) N-methyl-D-aspartate (NMDA) receptor antagonists, 2) ion channel antagonists, 3) alpha2-agonists, 4) nicotinic receptor agonists, 5) prostaglandin receptor antagonists, 6) adenosine agonists and adenosine kinase inhibitors, 7) neuropeptide antagonists, and 8) prosaposins. The results of preclinical and clinical trials are promising for these new agents. Whether these agents will be efficacious as single agents is yet to be determined; however, preliminary results show that combination therapy may be more beneficial with fewer side effects.
In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.
Two novel 16-substituted steroidal carboxylate esters derived from prednisolone, methyl 11 beta,17 alpha,21- trihydroxy-3,20-dioxo-pregna-1,4-diene-16 alpha-carboxylate (P16CM) and methyl 11 beta,21-dihydroxy-3,20-dioxo-pregna-1,4-diene-16 alpha- carboxylate (DeoxyP16CM) were evaluated for in vivo antiinflammatory and glucocorticoid activities. Results indicate that incorporation of a methoxycarbonyl group at the 16 position of prednisolone, as in P16CM, resulted in 5.5 times more local activity in the cotton pellet granuloma assay and 14 times more topical activity in the croton oil induced ear edema bioassay as compared with the parent compound prednisolone (P). The 17 alpha-dehydroxy analogue of P16CM (DeoxyP16CM) retained one-half the local activity of P in the cotton pellet granuloma bioassay and topical activity equal to P in the croton oil induced ear edema bioassay. Favorable dissociation of local from systemic effects is seen for these steroidal 16-carboxylate esters since their systemic antiinflammatory activity was significantly less than that of P, and their suppression of plasma corticosterone and ACTH levels was minimal. While P16CM does exhibit some thymolytic activity, DeoxyP16CM is essentially devoid of thymus atrophogenic effects at equiactive doses. Thus, these compounds may represent safer topical therapeutic agents.
Asialo-oligosaccharides obtained by sialidase digestion of asparagine-linked acidic sugar chains of promyelocytic leukemic cells (HL-60) differed in size distribution from those of cells induced to undergo myeloid or monocytoid differentiation. High-molecular-weight oligosaccharides which are predominant in HL-60 cells, decreased slightly during myeloid differentiation and markedly during monocytoid differentiation with concomitant increase of biantennary oligosaccharides. Structural analyses revealed that the induced monocytoid cells contain a series of complex-type oligosaccharides with bi-, tri-, and tetraantennary structures and high-molecular-weight oligosaccharides with N-acetyllactosamine repeating units, which show diversity in the presence or absence of the fucose residue linked to the reducing terminal N-acetylglucosamine, of the bisecting N-acetylglucosamine residue, and of the X-antigenic determinant in their outer chain moieties. Methylation analysis of each class of oligosaccharides of HL-60 cells and of their differentiated counterparts revealed the presence of similar heterogeneity in their structures, indicating that the difference is only quantitative. The results show that the shifts in size of the oligosaccharides found in the differentiated cells are derived from changes in outer chain formation and elongation of N-acetyllactosamine repeating units, and thus suggests that the decreased expression of N-acetylglucosaminyltransferases may be involved especially in the monocytoid differentiation program.
Five species of Mycobacterium were analyzed for the fatty acid composition of total and neutral lipids and of individual classes of phosphatides. The methyl esters of fatty acids were analyzed using the technique of gas liquid chromatography. It was found that myristic, palmitic, palmitoleic, and tuberculostearic acids were the major fatty acids present in all of the lipid components of these strains of tubercle bacilli. The ratio of unsaturated to saturated fatty acids is lowest in the most pathogenic strain, indicating a progressive increase in this ratio, and the highest ratio is represented by a saprophytic strain. An avian pathogenic organism falls in the center of this classification among the examined strains of mycobacteria. These results indicate that the ratio of unsaturated to saturated fatty acids of lipids can be used to determine and establish the pathogenic species of mycobacteria.
Michael-addition of 3-aminocrotonic acid ester 7 to aralkylidene acetoacetic acid esters 6 is followed by ring closure to give novel 4-aryl-1,4-dihydro-2,6-dimethyl-pyridine-3,5-dicarboxylates 8 with non-identical ester functions. In the series of 3-nitrophenyl derivatives (8, Ar=3-nitrophenyl) the pharmacological activities (coronary vasodilation, antihypertensive activity) of the asymmetrically substituted derivatives are shown to be superior to those of the corresponding symmetrically substituted derivatives in many cases. One representative of this class 3-ethyl-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (nitrendipine, Bay e 5009, No. 3) was selected for further development as an antihypertensive drug.
Endonuclease NaeI is a prototype for an unusual group of type II restriction endonucleases that must bind two DNA recognition sequences to cleave DNA. The naeIR gene, expressed from a Ptac promotor construct, was toxic to Escherichia coli in the absence of NaeI-sequence specific methylases. The naeIR gene was mutagenized with N-methyl-N'-nitrosoguanidine; four classes of NaeI variants were isolated in the absence of protecting methylase activity. Class I variants (T60I, E70K) lacked detectable cleavage activity, but displayed good sequence-specific DNA binding. Class II variants (D95N, G141D) displayed 1-5% of the wild-type cleavage activity and normal DNA binding. Class III variants (G131E, G131R, G155D, G245E) displayed significantly attenuated cleavage and binding activities. Class IV variants (G197D, G214R/A219T, G236S, L241P, G245E, G245R, G250E, G270E) lacked both cleavage and binding activities. These results imply two amino acids (Thr-60, Glu-70) essential for catalysis. In addition, two domains are indicated in NaeI: one (Thr-60 to Gly-155) mediates substrate binding and catalysis, the other (Gly-197 to Gly-270) may mediate binding of the activating DNA sequence. Our results are compared with the active site residues of EcoRI, EcoRV, and BamHI.