Short-chain Amides Research Articles

Accurate Identification of Periplasmic Urea-binding Proteins by Structure- and Genome Context-assisted Functional Analysis

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here.

Promoting Degradation of Polyamide-Microplastic Fibers Using Hydroxy Radical Generated by Electrochemical Advanced Oxidation Processes

Microplastic fibers (MPFs) generated by washing textile products made of synthetic fibers cannot be removed at sewage treatment plants and are released into rivers and oceans. In this study, we proposed using hydroxy radical (•OH) to degrade MPF. •OH exhibits strong oxidizing ability, allowing MPFs to be degraded. Herein, electrochemical advanced oxidation processes, which combines electrochemical oxygen reduction in water and UV irradiation, was used to generate •OH. In this paper, we investigated the degradation of polyamide 6/6 (PA6/6), as a common MPF. After 120 h degradation process, the sample thickness and fiber diameter decreased 32.0% and 13.7% respectively. After only 12 h, the sample toughness decreased 78.9%. Infrared spectroscopy confirmed that the signal of amide bonds (–CONH–) were reduced and new signal of carboxyl groups (–COOH) were formed in the sample after degradation. Gas chromatography confirmed that several short-chain amide compounds were released into the degraded solution. These results suggest that the amide bonds in the PA6/6 molecular structure are cleaved by the •OH, thereby accelerating the degradation of PA6/6. The proposed degradation method achieved a much faster degradation rate than other methods using microorganisms, and is expected to be a novel MPF degradation method.

Investigation of new ferrocenyl-artesunate derivatives as antiparasitics.

Artesunate (Ars) is a semisynthetic antimalarial drug and is a part of the artemisinin-based combination therapy arsenal employed for malaria treatment. The drug functions mainly by activation of its endoperoxide bridge leading to increased oxidative stress in malaria parasites. The purpose of this study was to ascertain the antiparasitic effects of combining ferrocene and Arsvia short or long chain ester or amide linkages (C1-C4). The compounds were evaluated for growth inhibition activity on the apicomplexan parasites, Plasmodium falciparum (P. falciparum) and Toxoplasma gondii (T. gondii). All the complexes demonstrated good activity against T. gondii with IC50 values in the low micromolar range (0.28-1.2 μM) and good to excellent antimalarial activity against a chloroquine sensitive strain of P. falciparum (NF54). Further investigations on T. gondii revealed that the likely mode of action (MoA) is through the generation of reactive oxygen species. Additionally, immunofluorescence microscopy suggested a novel change in the morphology of the parasite by complex C3, an artesunate-ferrocenyl ethyl amide complex. The complexes were not cytotoxic or showed low cytotoxicity to two normal cell lines tested.

Anticancer and antimicrobial effects of novel ciprofloxacin fatty acids conjugates

Ciprofloxacin (CP) has a confirmed cytotoxic action on some cancerous cells, but in high, non-pharmacological concentrations. Considering features of natural fatty acids, such as biocompatibility, biodegradability and their increased cellular uptake by cancer cells, it seems that combining them with a drug could improve its bioavailability, and thus cytotoxicity. Therefore, the aim of this study was coupling of CP with saturated and unsaturated fatty acids, and evaluation of their cytotoxicity, apoptosis-inducing effects and inhibition of IL-6 release in human primary (SW480) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3) and normal (HaCaT) cell lines. The PC3 cell line was the most sensitive to the presence of the obtained conjugates. The value of IC50 for oleic acid conjugate (4) was 7.7 μM, and it was 12 times lower than for CP alone (101.4 μM). The studied derivatives induced late apoptosis in all cancer cell lines, but not in normal cells. The most potent apoptosis inducer was conjugate 4, that resulted in the highest percentage of PC3 cells in late apoptosis (81.5% ± 3.9), followed by elaidic acid amide 5 (75% ± 4.8). The strongest pro-apoptic effects on SW480 cells were demonstrated by conjugates of DHA (8) and sorbic (2) acids, whereas in SW620 cell lines, compounds 2 and 5 appeared to be the most effective. To establish the mechanism of cytotoxic action of derivatives 2, 4, 5, the level of interleukin-6 (IL-6) was measured. The compounds with the highest cytotoxic potential significantly decreased the release of IL-6 by cancer cells. Additionally, all conjugates were evaluated for their in vitro antimicrobial activity. Short chain amides - crotonic (1) and sorbic (2) - were the most active against Staphyloccoci. The second-mentioned amide has shown both strong antistaphylococcal and antitumor properties.

Development of a flow injection analytical system for short chain amide determination based on a tubular bioreactor and an ammonium sensor.

Pseudomonas aeruginosa (P. aeruginosa) possesses intracellular amidase activity, which catalyses the hydrolysis of short aliphatic amides producing NH4+, and has already been used along with an ammonium ion selective electrode for amide quantification. However, the incorporation of a biological membrane turned to be a challenging process and either the final arrangement was prone to amidase losses or the recovery of the sensor coating after the interaction took too long. In this article a flow injection system with an ammonium acoustic wave sensor is proposed, and after testing several different arrangements for the biological element, the ultimate choice consisted of the immobilization of a P. aeruginosa cell-free extract in the inner wall of a tubular glass reactor, which resulted in a reliable analytical system. Response times less than one minute and complete recovery in less than two minutes assured conveniently fast analysis. The analytical system, as long as the column was properly stored in HEPES buffer containing 2 mM β-mercaptoethanol and 1 mM benzamidine and refrigerated when not in use, could be used at least for 20 working days, along a period of one month, maintaining the initial sensitivity.

Cage lifetimes of ionic liquids as studied by the magnetic field effect probe

Magnetic field effects (MFEs) on the photoinduced hydrogen abstraction reaction of benzophenone with phenol were investigated in ionic liquids (ILs) with a short alkyl chain (N,N,N-trimethyl-N-propylammonium bis(trifluoromethanesulfonyl)amide (TMPA TFSA)) and long alkyl chains ((N,N,N-trimethyl-N-octylammonium bis(trifluoromethanesulfonyl)amide (TMOA TFSA) and N-decyl-N,N,N-trimethylammonium bis(trifluoromethanesulfonyl)amide (DTMA TFSA)) by a nanosecond laser flash photolysis technique. In each ionic liquid, escaped radical yield of a benzophenone ketyl radical rapidly increased with increasing magnetic field strength (B) of 0 T < B≤ 0.01 T. At 0.01 T < B≤ 0.4 T, the escaped radical yield almost saturated in TMPA TFSA or gradually increased in TMOA TFSA and DTMA TFSA. At much higher fields of 0.4 T < B≤ 30 T, the yield gradually decreased, resulting in 10-15% decrease at 30 T. The observed MFEs can be explained by the hyperfine coupling and Δg mechanisms together with the relaxation mechanism. On the time profiles of the transient absorption observed for the benzophenone ketyl radical, MFEs were generated in the time range of 0 < t < 0.6 μs. The cage lifetimes of TMOA TFSA and DTMA TFSA were estimated to be at least 120 ns.

Differences of Urease Activity and Expression of Associated Genes According to Gastric Topography

We hypothesize that pH difference between acid-secreting corpus and non-secreting antrum might influence the activity of H. pylori's urease and/or related genes. We therefore measured urease activity and the expression of amiE whose encoded protein that hydrolyzes short-chain amides to produce ammonia. Fifty-four patients were recruited into this study. Each gastroscopic biopsy specimen collected from the antrum and body of each patient was immediately used to measure urease activity using serial changes of urease activity (ammonia levels) during 60 minutes. Probe specific for amiE was labeled with a biotin nick-translation kit and was used to detect expression of these genes (mRNA) in fresh-frozen gastroscopic biopsy specimens using fluorescent in situ hybridization (FISH). Urease activity at 60 minutes from the gastric antrum and body of all patients infected with H. pylori was 399.5 ± 490.5 and 837.9 ± 1038.9 μg/dL, respectively (p = .004). Urease activity in the antrum was correlated with H. pylori density. Urease activity or H. pylori density in the antrum was significantly correlated with chronic active inflammation; in contrast, this correlation was not found in the gastric body. The expression level of amiE was 1.5 times higher (p < .05) in the gastric body compared with the antrum. Topographically, the urease activity in body was much higher than in antrum. The expression level of amiE was higher in the gastric body compared with the antrum.

The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues.

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short-chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two-step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys-165 in AmiE and Cys-166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.

The effect of growth in continuous culture at various input C:N ratios on the expression of proteins involved in the transport and metabolism of methanol and short-chain amides byMethylophilus methylotrophus

Physiological regulation of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus was investigated following growth in continuous culture at various input C:N ratios. The concentrations of the methanol porin and methanol dehydrogenase were highest during C-limited growth (C:N 16.3). In contrast, the concentrations of the amide-urea porin, the amide-urea binding protein, formamidase and acetamidase (together with formamidase and acetamidase activities) were lowest during C-limited growth, but increased sharply as a function of the C:N ratio and were highest during dual CN-limited and N-limited growth (C:N 4.6–16.3). The results are discussed in terms of the physiological and biochemical requirements of growth at varying C:N ratios.

The effect of growth in continuous culture at various input C:N ratios on the expression of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus

Physiological regulation of proteins involved in the transport and metabolism of methanol and short-chain amides by Methylophilus methylotrophus was investigated following growth in continuous culture at various input C:N ratios. The concentrations of the methanol porin and methanol dehydrogenase were highest during C-limited growth (C:N<4.6), but declined gradually as a function of the increasing C:N ratio and were lowest during N-limited growth (C:N>16.3). In contrast, the concentrations of the amide-urea porin, the amide-urea binding protein, formamidase and acetamidase (together with formamidase and acetamidase activities) were lowest during C-limited growth, but increased sharply as a function of the C:N ratio and were highest during dual CN-limited and N-limited growth (C:N 4.6–16.3). The results are discussed in terms of the physiological and biochemical requirements of growth at varying C:N ratios.

Physiological regulation, purification and properties of urease fromMethylophilus methylotrophus

The methylotrophic bacterium Methylophilus methylotrophus hydrolyses urea to ammonia using a cytoplasmic urease (EC 3.5.1.5). During growth in continuous culture under various nutrient limitations, urease was induced by urea and short-chain amides (formamide and urea≫acetamide), and repressed by excess ammonia. The enzyme was purified using ammonium sulfate fractionation and fast protein liquid chromatography (FPLC), and exhibited a narrow substrate specificity (urea≫formamide and acetamide; no activity with other amides) with a Km for urea of 3.8 mM. Gel filtration FPLC and SDS-PAGE showed that the enzyme had a native Mr of approximately 190 000 and was composed of α (Mr 64 000), β (Mr 15 500) and γ (Mr 15 000) subunits in the probable ratio α2β2γ2. The physiological regulation and biochemical properties of the M. methylotrophus urease are compared with those of other bacterial ureases.

Physiological regulation, purification and properties of urease from Methylophilus methylotrophus

The methylotrophic bacterium Methylophilus methylotrophus hydrolyses urea to ammonia using a cytoplasmic urease (EC 3.5.1.5). During growth in continuous culture under various nutrient limitations, urease was induced by urea and short-chain amides (formamide and urea≫acetamide), and repressed by excess ammonia. The enzyme was purified using ammonium sulfate fractionation and fast protein liquid chromatography (FPLC), and exhibited a narrow substrate specificity (urea≫formamide and acetamide; no activity with other amides) with a K m for urea of 3.8 mM. Gel filtration FPLC and SDS-PAGE showed that the enzyme had a native M r of approximately 190 000 and was composed of α ( M r 64 000), β ( M r 15 500) and γ ( M r 15 000) subunits in the probable ratio α 2β 2γ 2. The physiological regulation and biochemical properties of the M. methylotrophus urease are compared with those of other bacterial ureases.

Characterisation of a binding‐protein‐dependent, active transport system for short‐chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus

Three genes (fmdCAB) encoding an outer-membrane porin for short-chain amides and urea, formamidase, and a putative regulatory protein in Methylophilus methylotrophus have previously been cloned and characterised. Three genes have now been identified downstream of fmdB, viz fmdD encoding a hydrophilic protein containing an N-terminal signal sequence, and fmdEF encoding hydrophobic transmembrane proteins. The derived amino acid sequence of mature FmdD (predicted molecular mass 41,870 Da) was similar to the cytoplasmic, amide-binding protein (AmiC) from Pseudomonas aeruginosa and to several periplasmic, solute-binding proteins from other bacteria. Mature FmdD was purified and shown to be a monomer (40-45 kDa) with the predicted N-terminal amino acid sequence (ADYPTA-). Equilibrium dialysis showed that the purified protein bound short-chain amides and urea with high affinity (Kd 7.2 microM for [14C]urea). SDS/PAGE and western blotting using antiserum to mature FmdD showed it was induced by short-chain amides and urea, and repressed by excess ammonia. The derived amino acid sequences of FmdE (32,822 Da) and FmdF (incomplete; >25,435 Da) were similar to the transmembrane proteins BraD/LivH and BraE/LivM, respectively, in various leucine/isoleucine/valine transport systems. Uptake of [14C]urea by washed cells was inhibited by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone and unlabelled formamide. It is concluded that FmdDEF comprise part of a high-affinity, binding-protein-dependent active-transport system for short-chain amides and urea in M. methylotrophus.

An outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus.

The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (M(r) 39204) showed that it was rich in beta-sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M(r) 40,000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.

Inhibition of red cell urea flux by anion exchange inhibitors

When they studied the chemical properties of red cell anion exchange inhibitors such as DIDS (4,4′-diisothiocyanate-2,2′-stilbene disulfonate), Barzilay et al. (1979) Membr. Biochem. 2, 227–254 also examined the benzene sulfonates. These molecules are structurally similar to half a DIDS molecule and are also specific anion exchange inhibitors with ID 50 values measured in mM, rather than μM, as for the stilbene disulfonates. We have studied several inhibitors of the benzene sulfonate (BS) class and found that they also inhibit red cell urea flux by up to 92% and stimulate water flux by up to 58%. The values of K inhib,app for urea flux inhibition are the same as the ID 50 values for anion flux inhibition; covalent DIDS completely suppresses the inhibition. These observations strongly suggest that the effect on urea flux is caused by BS binding at the stilbene site. Comparative studies on the short chain amides exclude lipid solubility and solute molar volume as factors that affect these BS actions. K stim,app for water flux stimulation is also related to the anion exchange ID 50 values; covalent DIDS suppresses the water flux stimulation. These observations on urea and water fluxes are consistent with a common driver, located at the stilbene site, which is responsible for the BS actions on urea, water and anion fluxes. The subsequent steps are independent with separate effectors to modulate each of the individual fluxes. These effectors are presumably located in different regions of the protein or proteins and carry out their separate processes by allosteric means.

Microbial degradation of acrylamide monomer

Acrylamide, a neurotoxic monomer with extensive industrial applications was found to be degraded by the microorganisms present in a tropical garden soil. A bacterium capable of degrading acrylamide was isolated from this soil by enrichment. It was found to be aerobic, gram-negative, motile, short rod and identified as Pseudomonas sp. The bacterium degraded high concentrations of acrylamide (4 g/l) to acrylic acid and ammonia which were utilized as sole carbon and nitrogen source for growth. An amidase was involved in the hydrolysis of acrylamide, which could act on other short chain amides like formamide and acetamide but not on acrylamide analogues: methacrylamide and N,N-methylene bisacrylamide. The enzyme was sensitive to catabolite repression by succinate both in presence as well as absence of nitrogen source.

ChemInform Abstract: SYNTHETIC LUTEINIZING HORMONE RELEASING FACTOR ANALOGS, SERIES OF SHORT‐CHAIN AMIDE LRF HOMOLOGS CONVERGING TO THE AMINO TERMINUS

Chemischer InformationsdienstVolume 4, Issue 38 Natural Products ChemInform Abstract: SYNTHETIC LUTEINIZING HORMONE RELEASING FACTOR ANALOGS, SERIES OF SHORT-CHAIN AMIDE LRF HOMOLOGS CONVERGING TO THE AMINO TERMINUS J. RIVIER, J. RIVIERSearch for more papers by this authorW. VALE, W. VALESearch for more papers by this authorR. BURGUS, R. BURGUSSearch for more papers by this authorN. LING, N. LINGSearch for more papers by this authorM. AMOSS, M. AMOSSSearch for more papers by this authorR. BLACKWELL, R. BLACKWELLSearch for more papers by this authorR. GUILLEMIN, R. GUILLEMINSearch for more papers by this author J. RIVIER, J. RIVIERSearch for more papers by this authorW. VALE, W. VALESearch for more papers by this authorR. BURGUS, R. BURGUSSearch for more papers by this authorN. LING, N. LINGSearch for more papers by this authorM. AMOSS, M. AMOSSSearch for more papers by this authorR. BLACKWELL, R. BLACKWELLSearch for more papers by this authorR. GUILLEMIN, R. GUILLEMINSearch for more papers by this author First published: September 18, 1973 https://doi.org/10.1002/chin.197338400Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume4, Issue38September 18, 1973 RelatedInformation

Synthetic luteinizing hormone-releasing factor analogs. Series of short-chain amide LRF homologs converging to the amino terminus.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthetic luteinizing hormone releasing factor analogs. Series of short-chain amide LRF homologs converging to the amino terminusJ. Rivier, W. Vale, R. Burgus, N. Ling, M. Amoss, R. Blackwell, and R. GuilleminCite this: J. Med. Chem. 1973, 16, 5, 545–549Publication Date (Print):May 1, 1973Publication History Published online1 May 2002Published inissue 1 May 1973https://doi.org/10.1021/jm00263a031RIGHTS & PERMISSIONSArticle Views65Altmetric-Citations56LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (735 KB) Get e-Alerts Get e-Alerts