Metabolism of 6-aminonicotinic acid in Escherichia coli.

A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-14C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6-aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.     

Metabolism of 6-Mercaptopurine by Resistant Escherichia coli Cells

Coggin, Joseph H. (University of Chicago, Chicago, Ill.), Muriel Loosemore, and William R. Martin. Metabolism of 6-mercaptopurine by resistant Escherichia coli cells. J. Bacteriol. 92:446-454. 1966.-6-Mercaptopurine (MP) utilization as a source of purine in MP-sensitive and -resistant cultures of Escherichia coli was investigated. The label of MP-8-C(14) appeared in adenine and guanine of ribonucleic acid and deoxyribonucleic acid in sensitive and resistant cultures. Studies using MP-S(35) further demonstrated that the MP moiety was degraded, as shown by a rapid decrease in radioactivity from cells upon exposure to MP for 20 min. Enzymatic analysis showed that MP was converted to 6-mercaptopurine ribonucleotide (MPRP) by extracts derived from both sensitive and resistant cells. Resistant cell preparations, however, degraded MPRP to inosine monophosphate (IMP) rapidly when compared with analogue degradation by sensitive cells. Inosineguanosine-5'-phosphate pyrophosphorylase from resistant cells did not catalyze the synthesis of IMP from hypoxanthine when the cells were cultured in the presence of MP, but these enzyme preparations actively converted guanine to guanosine monophosphate (GMP). Pyrophosphorylase derived from resistant cells cultured in medium without MP catalyzed the conversion of hypoxanthine to IMP and also guanine to GMP. These observations suggest that inosine-guanosine-5'-phosphate pyrophosphorylase is composed of two distinct enzymes. The mode of resistance to MP in E. coli is related to an enhancement of the enzymatic degradation of MPRP to the pivotal purine intermediate, IMP.     

Metabolism of a proline analogue, l-thiazolidine-4-carboxylic acid, by Escherichia coli

Unger, Leon (University of Illinois, Urbana), and R. D. DeMoss. Metabolism of a proline analogue, l-thiazolidine-4-carboxylic acid, by Escherichia coli. J. Bacteriol. 91:1564-1569. 1966.-Resting cells of Escherichia coli K-12, pregrown in a proline- and thioproline-free medium, oxidize the proline analogue, l-thiazolidine-4-carboxylic acid (l-thioproline), without a lag with the consumption of 1 atom of oxygen per mole of thioproline. The organism also oxidizes cysteine and formaldehyde, the chemical precursors of thioproline. The total oxygen consumed is the same whether the substrate is thioproline, cysteine, formaldehyde, or an equimolar mixture of cysteine and formaldehyde. The results suggest that neither cysteine nor formaldehyde are free intermediates in the oxidative pathway. Thioproline is available as a metabolic carbon source for the synthesis of the ribonucleic acid bases, guanine and uracil.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

Metabolism of [6]-gingerol in rats

The metabolic fate of [6]-gingerol, one of the active constituents of Zingiber officinale Roscoe, was investigated using rats. The bile of rats orally administered [6]-gingerol was shown to contain a major metabolite (1) by HPLC analysis. Although the metabolites derived from [6]-gingerol were not detected in the urine, the ethyl acetate extract of the urine after enzymatic hydrolysis was shown to contain six minor metabolites (2-7). Their structures were determined to be (S)-[6]-gingerol-4'-O-beta-glucuronide (1), vanillic acid (2), ferulic acid (3), (S)-(+)-4-hydroxy-6-oxo-8-(4-hydroxy-3-methoxyphenyl) octanoic acid (4), 4-(4-hydroxy-3-methoxyphenyl)butanoic acid (5), 9-hydroxy [6]-gingerol (6) and (S)-(+)-[6]-gingerol (7) based on spectroscopic and chemical data. The total cumulative amount of 1 excreted in the bile and 2-7 in the urine during 60 h after the oral administration of [6]-gingerol were approximately 48% and 16% of the dose, respectively. The excretion of 2-7 in the urine decreased after gut sterilization. On the other hand, the incubations of [6]-gingerol with rat liver showed the presence of 9-hydroxy [6]-gingerol, gingerdiol (8), and (S)-[6]-gingerol-4'-O-beta-glucuronide (1). These findings suggest that the gut flora and enzymes in the liver play an important part in the metabolism of [6]-gingerol.     

Synthesis, characterization, DNA-binding and photocleavage of complexes [Ru(phen)2(6-OH-dppz)]2+ and [Ru(phen)2(6-NO2-dppz)]2+

Two new complexes, ([Ru(phen)(2)(6-OH-dppz)](2+)) (1) and ([Ru(phen)(2)(6-NO(2)-dppz)](2+)) (2) (phen=1,10-phenanthroline; 6-OH-dppz=6-hydroxyl-dipyrido[3,2-a:2',3'-c]phenazine; 6-NO(2)-dppz=6-nitro-dipyrido[3,2-a:2',3'-c]phenazine), have been synthesized and characterized by elemental analysis, ES-MS (electrospray mass spectra), (1)H NMR, UV-Vis (UV-visible) and CV (cyclic voltammetry). The DNA-binding behaviors of both complexes have been studied by spectroscopic methods and viscosity measurements. The results indicate that the two complexes all bind to calf thymus DNA (CT-DNA) in an intercalative mode, and the DNA-binding affinity of complex 2 is greater than that of complex 1. In addition, complex 1 can promote photocleavage of pBR322 DNA upon irradiation, whereas complex 2 can promote cleavage of pBR322 DNA both upon irradiation and in the dark, with more efficient cleavage occurring upon irradiation. Theoretical studies for these complexes have been also carried out with the density functional theory (DFT) method. The difference in the DNA-binding behaviors of the two complexes can be reasonably explained by the DFT calculations.     

Metabolism of glutamic acid in a mutant of Escherichia coli

Vender, Joyce (Indiana University, Bloomington), Kunthala Jayaraman, and H. V. Rickenberg. Metabolism of glutamic acid in a mutant of Escherichia coli. J. Bacteriol. 90:1304-1307. 1965.-A mutant strain of Escherichia coli W1485 was selected for its ability to utilize glutamic acid as the sole source of carbon. Growth of the mutant on glutamic acid led to the repression of glutamic acid dehydrogenase formation. The mutant differed from the wild-type strain in that glutamic decarboxylase activity was absent from the mutant under conditions of growth which supported the formation of this enzyme in the parent strain. Evidence is presented which suggests that loss of the decarboxylase activity results in the acquisition of the ability to utilize glutamic acid as sole source of carbon; a pathway of glutamate utilization via transamination is proposed.     

N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea and N'-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]-thiourea as potent inhibitors of multidrug-resistant human immunodeficiency virus-1.

We have replaced the pyridyl ring of trovirdine with an alicyclic cyclohexenyl, adamantyl or cis-myrtanyl ring. Only the cyclohexenyl-containing thiourea compound N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-bromopyridyl)]- thiourea (HI-346) (as well as its chlorine-substituted derivative N-[2-(1-cyclohexenyl)ethyl]-N'-[2-(5-chloropyridyl)]- thiourea/HI-445) showed RT inhibitory activity. HI-346 and HI-445 effectively inhibited recombinant RT with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cell-free RT inhibition assays was: HI-346 (IC50 = 0.4 microM) > HI-445 (IC50 = 0.5 microM) > trovirdine (IC50 = 0.8 microM) > MKC-442 (IC5 = 0.8 microM) = delavirdine (IC50 = 1.5 microM) > nevirapine (IC50 = 23 microM). In accord with this data, both compounds inhibited the replication of the drug-sensitive HIV-1 strain HTLV(IIIB) with better IC50 values than other anti-HIV agents tested. The ranking order of efficacy in cellular HIV-1 inhibition assays was: HI-445 = HI-346 (IC50 = 3 nM) > MKC-442 (IC50 = 4 nM) = AZT (IC50 = 4 nM) > trovirdine (IC50 = 7 nM) > delavirdine (IC50 = 9 nM) > nevirapine (IC50 = 34 nM). Surprisingly, the lead compounds HI-346 and HI-445 were 3-times more effective against the multidrug resistant HIV-1 strain RT-MDR with a V106A mutation (as well as additional mutations involving the RT residues 74V,41L, and 215Y) than they were against HTLV(IIIB) with wild-type RT. HI-346 and HI-445 were 20-times more potent than trovirdine, 200-times more potent than AZT, 300-times more potent than MKC-442, 400-times more potent than delavirdine, and 5000-times more potent than nevirapine against the multidrug resistant HIV-1 strain RT-MDR. HI-445 was also tested against the RT Y181C mutant A17 strain of HIV-1 and found to be >7-fold more effective than trovirdine and >1,400-fold more effective than nevirapine or delavirdine. Similarly, both HI-346 and HI-445 were more effective than trovirdine, nevirapine, and delavirdine against the problematic NNI-resistant HIV-1 strain A17-variant with both Y181C and K103N mutations in RT, although their activity was markedly reduced against this strain. Neither compound exhibited significant cytotoxicity at effective concentrations (CC50 >100 microM). These findings establish the lead compounds HI-346 and HI-445 as potent inhibitors of drug-sensitive as well as multidrug-resistant stains of HIV-1.     

Differential genotoxic effects of antitumor trans-[PtCl(2)(E-iminoether)(2)] and cisplatin in Escherichia coli

In the present work, we measured survival and the platinum on the genome after treatment of repair-proficient or repair-deficient Escherichia coli strains with trans-[PtCl₂(E-iminoether)₂] and compared these results with the effects of “classical” cisplatin. We found that toxicity of antitumor trans-[PtCl₂(E-iminoether)₂] in repair-deficient trains was much less than that of cisplatin. This markedly reduced toxicity was not a consequence of the reduced uptake or low levels of DNA binding in the bacteria cells but rather appeared to reflect DNA binding mode of this trans-platinum drug different from that of cisplatin.     

(2S,3S)-Oxirane-2,3-dicarboxylic acid: a privileged platform for probing human cysteine cathepsins

The notion that human cysteine cathepsins contribute only to general protein turnover within the lysosomes has changed in the last decade in a substantial manner. A continuously growing number of data accumulated in different fields of life sciences revealed that these enzymes are involved in a variety of pivotal physiological processes. To investigate these particular fraction of proteolytical activity of the human degradome even in a complex cellular environment, chemical probes that covalently label the corresponding proteases proved to be versatile tools. (2S,3S)-Oxirane-2,3-dicarboxylic acid provides an ideal platform for the design of such probing systems. Depending on the complexity of the attached recognition elements, either the activity of the entire group of human cysteine cathepsins or individual members can be detected.     

The glutamate-dependent acid resistance system of Escherichia coli and Shigella flexneri is inhibited in vitro by L-trans-pyrrolidine-2,4-dicarboxylic acid

Strains of Escherichia coli K-12, O157:H7, and Shigella flexneri grown to stationary phase in complex unbuffered media can survive for several hours at pH 2.5. This stationary-phase acid resistance phenotype is dependent upon the alternate sigma factor sigmas and the supplementation of either glutamate or glutamine in the acidified media used for acid challenge. Acid resistance under these defined conditions can be inhibited by the glutamate analog L-trans-pyrrolidine-2,4-dicarboxylic acid which blocks uptake of glutamate/glutamine by selective inhibition. The gadC gene, encoding an inner membrane antiporter essential for the expression of acid resistance, could not be detected in other family members of the Enterobacteriacae.