Screening of Microorganisms Producing 5-Amino-4-imidazole-carboxamide and D-Ribose from 5-Amino-4-imidazole-carboxamide-riboside

Rice has evolved metabolic and morphological adaptations to low-oxygen stress to grow in submerged paddy fields. To characterize the molecular components that mediate the response to hypoxia in rice, we identified low-oxygen stress early response genes by microarray analysis. Among the highly responsive genes, five genes, OsHREF1 to OsHREF5, shared strong homology. They encoded small proteins harboring two EF-hands, typical Ca²⁺-binding motifs. Homologous genes were found in many land plants, including SlHREF in tomato, which is also strongly induced by hypoxia. SlHREF induction was detected in both roots and shoots of tomato plants under hypoxia. With the exception of OsHREF5, OsHREF expression was unaffected by drought, salinity, cold, or osmotic stress. Fluorescent signals of green fluorescent protein-fused OsHREFs were detected in the cytosol and nucleus. Ruthenium red, an inhibitor of intracellular Ca²⁺ release, repressed induction of OsHREF1-4 under hypoxia. The HREFs may be related to the Ca²⁺ response to hypoxia.     

5-Amino-4-imidazolecarboxamide riboside (Z-riboside) metabolism in eukaryotic cells

Metabolites of 5-amino-4-imidazolecarboxamide riboside (Z-riboside) have potential roles in the regulation of cellular metabolism and as pharmacological agents in several pathological situations. Before studying Z-riboside metabolism it was necessary to develop methods for identifying and quantitating 5(4)-amino-4(5)-imidazolecarboxamide metabolites. These studies utilized Chinese hamster ovary fibroblast auxotrophic mutants to identify and isolate compounds relevant to Z-riboside metabolism by a combination of high performance liquid chromatographic procedures. In order to study Z-riboside metabolism wild-type and mutant cells were cultured in Z-riboside. This ribosyl precursor to a purine de novo intermediate does not undergo any detectable phosphorolysis but rather is phosphorylated by adenosine kinase in an unregulated manner. This results in the intracellular accumulation of 5-amino-4-imidazolecarboxamide ribotide (ZMP), the levels of which control flow from Z-riboside to the following metabolites: 1) IMP and other purine nucleotides, 2) 5-amino-4-imidazole-N-succinocarboxamide ribotide (sZMP), and 3) 5-amino-4-imidazolecarboxamide riboside 5'-triphosphate (ZTP). At low ZMP concentrations, the predominant metabolic fate is IMP. Initially, IMP enters the adenylate and guanylate pools, but subsequently is hydrolyzed to inosine and this phosphorolyzed to hypoxanthine. At intermediate ZMP concentrations there is net retrograde flux through the bifunctional enzyme adenylosuccinate AMP lyase resulting in sZMP synthesis and antegrade flux leads to the accumulation of adenylosuccinate. At high ZMP concentrations, ZTP accumulates. In addition to these effects on purine metabolism, pyrimidine nucleotide pools are depleted when ZMP accumulates. These results are discussed in relation to the regulation of purine nucleotide synthesis and the use of Z-riboside as a pharmacological intervention in pathophysiological situations.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide Riboside by Escherichia coli

The acculnulation of 5 (4) -amino-4 (5) -imidazolecarboxamide riboside (AICA-R) in the culture medium of sulfonamide-inhibited Escherichia coli, and E. coli-like bacteria was studied. E. coli strain Band 32 strains of E. coli-like bacteria accumulated more than 50 μmoles of AICA-R in test tube scale experiments, and one of E. coli-like bacteria accumulated 358 μmoles. E. coli B-96 (purine-requiring mutant) had ability to accumulate AICA-R in the glucose-salt medium containing purine bases, especially xanthine. The addition of glycine alone or together with glutamic acid to the glucose-salt medium increased the accumulation of AICA-R by sulfadiazine-inhibited E. coli strain B. The accumulation was considerably increased by the addition of polypeptone or casein hydrolysate.

AICA-R accumulated during sulfadiazine bacteriostasis of E. coli strain B was purified and crystallized according to the procedure of Greenberg and Spilman, and light amber colored crystals were obtained.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazolecarboxamide by Escherichia coli in a Shaking Culture

In the previous paper the author reported that 5(4)-amino-4(5) -imidazolecarboxamide (AICA) was accumulated in peptone medium by Escherichia coli strain B grown as a shaking culture in the absence of sulfonamide inhibitor and glucose. It appeared from the further investigations that l-tryptophan would not replace peptone for the accumulation. However, it was found that indole produced from l-tryptophan by E. coli gave pink color by the Bratton nad Marshall method. In order to eliminate the effect of indole, the procedure by petroleum-ether treatment was applied and it was ascertained that E. coli had an ability to accumulate AICA and AICA-riboside in the peptone or L-tryptophan medium without glucose and sulfonamide inhibitor. But the concentrations of AICA and AICA-riboside measured by the above procedure were smaller than those determined by the Bratton and Marshall method which measured indole produced by the bacteria at the same time.     

Studies on the Accumulation of 5(4)-Amino-4(5)-imidazole- carboxamide by Escherichia coli in a Shaking Culture

Various attempts were made to accumulate 5 (4) -amino-4 (5) -imidazolecarboxamide (abreviated as AICA; in this paper, separate analyses for the riboside forms were not attempted, and hence AICA and AICA-riboside will be presented as total AICA) in a shaking culture medium by Escherichia coli strain B. The accumulation of non-acetylatable, diazotizable amines was accomplished by the addition of 0.01% of sulfadiazine, 0.2% of glucose, and 2% of peptone in the medium for sixteen hours at 30°C. E coli strain B was able to accumulate the amines in the pepton medium, even when glucose and the sulfonamide inhibtor were omitted. Although paper chromatographic and spectrophotometric analyses proved the accumulation of AICA and AICA-riboside by E. coli train B in the medium, another substance colored by the Bratton and Marshall method was also accumulated.     

Meiosis activating sterols (MAS) and fertility in mammals and man.

In mammals two meiosis activating sterols (MAS) have been found to activate meiotic resumption in mouse oocytes, in vitro. FF-MAS (4, 4-dimethyl-5alpha-cholesta-8,14,24-triene-3beta-ol) was extracted from human preovulatory follicular fluid and T-MAS (4, 4-dimethyl-5alpha-cholest-8,24-diene-3beta-ol) from bull testicular tissue. Quite unexpected, these two sterols, which introduce the cholesterol biosynthetic pathway from lanosterol, may be locally acting substances with important physiological function for reproduction. FF-MAS and T-MAS are present in the preovulatory follicular fluid of different mammalian species and have the capacity to initiate resumption of meiosis in mouse oocyte cultured in the presence of hypoxanthine, a natural meiosis maturation inhibitor. FF-MAS is produced by the cumulus cells of intact oocyte-cumulus complexes upon FSH-stimulation and provides the oocyte with a go-signal for the resumption of meiosis. T-MAS constitutes the vast majority of MAS found in the mammalian testis and in the human ejaculate; in particular a high concentration is found in the spermatozoa. T-MAS may be produced by the spermatids and the presence of T-MAS in spermatozoa may suggest that T-MAS plays a role in fertilization by affecting the second meiotic division.J. Exp. Zool. (Mol. Dev. Evol.) 285:237-242, 1999.     

4-Amino-6-piperazin-1-yl-pyrimidine-5-carbaldehyde oximes as potent FLT-3 inhibitors

A series of 4-amino-6-piperazin-1-yl-pyrimidine-5-carbaldehyde oximes has been discovered and developed as potent FLT3 tyrosine kinase inhibitors. The series exhibited potent antiproliferative activity against both an FLT3 ITD-mutated human leukemic cell line as well as a wild-type FLT3 BaF(3) expressed cell line. The structure-activity relationship of this class of compounds is described.     

3-Amino-2(5H)furanones as inhibitors of subgenomic hepatitis C virus RNA replication

A new class of compounds able to block the replication of subgenomic HCV RNA in liver cells is described. 3-Amino-2(5H)furanones 4 may be regarded as diketoacid analogues and were obtained by basic rearrangement of the isoxazolidine nucleus.     

Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

7-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose. A constituent of the endotoxin of Bordetella pertussis.

Hydrolysis of the Bordetella pertussis endotoxin, extracted from both "phase I" and "phase IV" bacteria, with 4 M HCl for 1 h at 100 degrees C, released the disaccharide named in the title; it was isolated by paper electrophoresis or by ion-exchange chromatography in about 1% yield (w/w). The structure of the heptose could be rigorously established by chemical degradation; the facts that the glucosaminidic linkage was hydrolysed by an enzyme preparation containing both, alpha and beta-N-acetylglucosaminidase activities, whereas it was resistant to cleavage by pure beta-N-acetylglucosaminidase strongly support the assumption that the disaccharide contains an alpha-D-glucosaminide linkage.     

5-Amino-4-imidazolecarboxamide is a mutagen in E. coli

5-Amino-4-imidazolecarboxamide (5A4IC), the base moiety of a common intermediate in de novo purine biosynthesis, was found to be mutagenic in E. coli. Using a series of mutants in the tryptophan synthetase A gene, 5A4IC was observed to cause transition and transversion mutations at similar levels. At 400 micrograms/ml in the growth medium, it stimulates the base substitution GC----AT 4.8-fold; AT----GC 20-fold; AT----CG (2 sites) an average of 6.0-fold; AT----TA 7.8-fold; and GC----CG 6.1-fold. The transversion GC----TA was not tested. In contrast to the base, 5-amino-4-imidazolecarboxamide riboside is not mutagenic at a similar molar concentration.