Catechol-based inhibitors of bacterial urease

Targeted covalent inhibitors of urease were developed on the basis of the catechol structure. Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC₅₀?=?518?nM and$k_{\mathit{inact}}/K_i$?=?1379?M^?1?s^?1. Inhibitory activity of this compound was better and toxicity lower than those obtained for the starting compound – catechol. The molecular modelling studies revealed a mode of binding consistent with structure-activity relationships.     

The structure of rooster-comb dermatan sulfate. Fragmentation of the polysaccharide chains by chondroitinase AC-II digestion,and by periodate oxidation,followed by alkali cleavage

The polysaccharide-chain fragments of rooster-comb dermatan sulfates (RC-20 and RC-30) were obtained by chondroitinase AC-II digestion and by periodate oxidation, followed by alkaline cleavage, and their structures analyzed both quantitatively and qualitatively. RC-20 having a lower d-glucuronic acid content (22.6%) is composed preponderantly of large clusters of N-acetyldermosine sulfate (M_r～17 600–41 000) at the nonreducing terminal, whereas RC-30, having a higher d-glucuronic acid content, (41.4%) is poor in this cluster. Both RC-20 and RC-30 have an N-acetyldermosine sulfate cluster (M_r 6500–7300) within the polysaccharide chains. Most N-acetylchondrosine sulfate units of RC-20 and RC-30 exist as clusters, the large clusters (M_r～17 600) being preponderant in RC-30; both RC-20 and RC-30 contain a large proportion of N-acetylchondrosine sulfate clusters (M_r 3500 and 9000) that corresponds to the uronic acid content. In RC-30, most N-acetyldermosine disulfate units (13.4%) are linked to N-acetylchondrosine sulfate units or clusters.     

Design,synthesis, molecular docking,anti-Proteus mirabilis and urease inhibition of new fluoroquinolone carboxylic acid derivatives

New hydroxamic acid, hydrazide and amide derivatives of ciprofloxacin in addition to their analogues of levofloxacin were prepared and identified by different spectroscopic techniques. Some of the prepared compounds revealed good activity against the urease splitting bacteria, Proteus mirabilis. The urease inhibitory activity was investigated using indophenol method. Most of the tested compounds showed better activity than the reference acetohydroxamic acid (AHA). The ciprofloxacin hydrazide derivative 3a and levofloxacin hydroxamic acid 7 experienced the highest activity (IC₅₀ = 1.22 μM and 2.20 μM, respectively). Molecular docking study revealed high spontaneous binding ability of the tested compounds to the active site of urease.     

Taxonomic analysis of volatiles emitted by ornamental crabapple flowers

To identify and classify volatile compounds in the fragrances of crabapple flowers from ornamental and original species in the peak April Tai’an, Shandong flowering season. Volatile components from flowers were identified using dynamic-headspace sampling, purge and trap and GC/MS. Eighty compounds were detected in the 17 ornamental crabapple cultivars, while sixty-eight compounds were found in the nine original species, including forty-four compounds in both the original species and cultivars. The main volatile components and common components of crabapple flower aroma include: 3-methyl-1-butanol, ocimene, benzyl alcohol, 3-methyl-4-oxo-pentanoic acid and heptane. Based on the relative volatile content the 26 taxa could be classified into seven different groups through UPGMA cluster analysis. The different volatile contents, such as myrcene and benzaldehyde, result in the extraordinary aroma of different crabapple varieties. M. ‘Dolgo’, M. ‘Eleyi’, M. ‘Hopa’, M. ‘Liset’, M. ‘Makamik’ and M. ‘Royalty’ are good breeding varieties of sweet flowering crabapples which could be used to breed additional ornamental cultivars with excellent fragrance and high value.     

Microenvironmental manipulation of the observed michaelis constant of ribulose diphosphate carboxylase

Urease and ribulose 1, 5-diphosphate carboxylase can be bound to Sepharose to give an immobilized two-enzyme system which catalyzes the reaction urea → H₂CO₃ → phosphoglyceric acid. The observed Km of the system for urea approaches the lower value for urease when carboxylase levels on the gel exceed urease levels. If a similar system operates in the chloroplast, the high Km (H₂CO₃) of ribulose 1,5-diphosphate carboxylase may not be metabolically significant.     

Competition by 4-chloronitrosobenzene for the active glycolaldehyde intermediate of transketolase

The incubation of 4-chloronitrosobenzene with yeast transketolase, Mg²⁺, and thiamime pyrophosphate in the presence of excess xylulose-5-phosphate resulted in the formation of N-(4-chlorophenyl)glycolhydroxamic acid. This enzyme-catalyzed C₂ transfer displayed a K_m of 0.92 mM and a V_max of 5.2 × 10^?2 μmol min^?1 unit enzyme^?1. Conversion was inhibited by the normal acceptor sugar, ribose-5-phosphate, with a K_i of 0.35 mM. Kinetic analysis showed inhibition was competitive in nature, reinforcing the proposed theory for similarity in catalytic formation of both the hydroxamic acid and sedoheptulose-7-phosphate. Most interesting about the conversion of this alternative substrate is that even at high concentrations of ribose-5-phosphate, a significant amount of the nitroso compound was converted to the hydroxamic acid, implying that 4-chloronitrosobenzene can successfully compete for active glycoaldehyde. Using the yeast enzyme as a model for transketolase in higher organisms, the adventitious conversion of such xenobiotics in vivo is proposed.     

Stimulation of human T-lymphocyte chemokinesis by arachidonic acid.

The migration of human T lymphocytes, assessed in modified Boyden chambers, was chemokinetically stimulated by arachidonic acid in a dose-related manner that achieved a peak level of 127 ± 34% enhancement (mean ± SD) at 8 μM arachidonic acid. The chemokinetic effect was dependent on the metabolism of the arachidonic acid by the T lymphocytes as derivatives of arachidonic acid that do not serve as prostaglandin and thromboxane precursors were without effect, while the cyclo-oxygenase inhibitors indomethacin (ID₅₀ = 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA) (ID₅₀ = 20 μM) suppressed the stimulation of migration by arachidonic acid. The cyclo-oxygenase product 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) reproduced part of the chemokinetic effect of arachidonic acid, but the lipoxygenase product 12-l-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) as well as PGE₂, PGF_2α, and thromboxane B₂ had no stimulatory activity. The ability of ETYA, but not indomethacin, to suppress the migration of unstimulated T lymphocytes suggested that a lipoxygenase metabolite of endogenous arachidonic acid contributes to the maintenance of their normal levels of spontaneous migration.     

Urease from leaves of Glycine max and Zea mays

Extracts of Glycine max and Zea mays leaves catalysed the release of ¹⁴CO₂ from [¹⁴C] urea with multiple pH maxima (5.5 and 9.0 for G. max; 5.5, 7.5 and 8.8 for Z. mays). Evidence was obtained that the principal activities, at pH 5.5 and 8.8–9.0 catalysed the same reaction stoichiometry as did urease purified from jackbean seeds (EC 3.5.1.5). The urease activities with these pH optima were not resolved by ammonium sulphate fractionation, DEAE-cellulose chromatography, or gel filtration chromatography. Many structural analogues of urea inhibited leaf urease, the most effective being amino acid hydroxamates, hydroxyurea and selenourea. Allantoic acid and ureidoglycolate are probably not alternative substrates because they showed at most only weak competitive inhibition with respect to radioactive urea.     

Inducible Formation of Urease in Canavalia ensiformis

Increase in urease activity in leaves of Canavdlia ensifomis has been demonstrated. The activity of excised leaves increased by about 100 percent when 1.5 × 10^?1M urea was added externally as inducer. Glycine-1-¹⁴C was used to investigate whether the increase in activity was dependent on de novo protein synthesis. The Incorporation of labeled amino acid into urease was twofold higher in induced samples than in non-induced ones. This indicates that the increase in activity is connected with de nova protein synthesis. The once increased activity was always followed by a rapid decrease. The urease activity was lost constantly with time after the external addition of ammonia in vivo. The inhibitory action of ammonia on urease fa vitro was eliminated by dialysis. Accordingly it may be concluded that the loss of activity was dependent on the product repression by ammonia.     

Resistance to acetohydroxamate acquired by slow adaptive increases in urease in cultured tobacco cells

Urease activity of tobacco XD cells (1U cells) had undergone a 4-fold increase (4U cells) during a year of growth on urea (Skokut and Filner 1980 Plant Phvsiol 65: 995-1003). A clone of 4U cells gave rise to 12U cells during another year of growth on urea. The doubling time of 12U cells on urea is 2.2 days, compared to about 4 days for 1U cells, while 1U and 12U cells double in 2 days on nitrate. Acetohydroxamic acid (AHA), a specific inhibitor/reversible inactivator of jack bean urease, affects tobacco cell urease similarly. Fifty per cent inhibition of growth by AHA occurred at 20 micromolar in 1U cells growing on urea and at 165 micromolar in 12U cells growing on urea, but at 600 micromolar for either 1U or 12U cells growing on nitrate. When 12U cells were grown on urea with 100 micromolar AHA, extractable urease activity decreased 80% within 2.5 hours and remained at this level for 2 weeks; the doubling time increased to 3.7 days, and intracellular urea rose 2-fold, compared to 12U cells grown on urea without AHA. Urease of 12U cells inactivated by AHA in vivo could be reactivated to its pre-AHA level by incubation at 30 C after extraction and separation from free AHA. AHA inhibited incorporation of ¹⁵N from [¹⁵N]urea into Kjeldahl nitrogen in the cells, in spite of the increased intracellular urea. These results indicate that AHA acts primarily by inhibiting urease action, rather than by inhibition of formation of urease protein or of uptake of urea. Because 12U cells are 8 times more tolerant of AHA than 1U cells, it is likely that growth on urea in the presence of AHA should select strongly for cells with high urease.     

The self-association of jack bean urease and its modification by silver ions.

At low ionic strength urease has been found to dissociate at protein concentrations below 1 × 10⁸m. The inhibition of enzyme activity by Ag⁺ has been used to demonstrate this. The inhibition by Ag⁺ has been shown to be independent of dissociation but, at dilutions where dissociation occurs, silver ion modifies the process. Urease is aggregated by Ag⁺ at high Ag⁺:protein ratios. Such inactive aggregates can be solubilized and reactivated by dithiothreitol. Further evidence has been obtained indicating the similarity of the (8n) and (16n) forms of urease. The phenomena of inhibition and aggregation in the presence of the heavy metal ion have been shown to be separate processes.     

In vitro proteolytic processing of pea and jack bean storage proteins by an endopeptidase from lupin seeds

The highly specific proteolytic breakdown observed upon prolonged treatment of pea legumin and pea and jack bean vicilin with a thiol endopeptidase purified from mature lupin seeds has been studied in detail. Proteolytic cleavage occurred in the acidic subunits of pea legumin, whereas the basic subunits were unaffected. Jack bean vicilin (M, 47 K) was cleaved near the middle of the polypeptide chain, whereas pea vicilin (M, 50 K) was cleaved into two fragments of M, 30 K and 20 K, respectively. The 30 K M, polypeptide chain contained covalently linked carbohydrate and had an N-terminal sequence suggesting that cleavage had taken place between the α and β region of the vicilin 50 K M, polypeptide as previously described in vivo. These results suggested that the cleavage specificity of lupin endopeptidase was in the proximity of paired arginine amino acid residues.The changes in the vicilin polypeptides due to proteolytic cleavage by lupin enzyme and those occurring during germination of pea seeds are also reported and discussed.     

Kinetic properties, and location of nonspecific phosphomonoesterases in subcellular fractions of fasciola hepatica

Optimal activity was recorded at pH 4.5–5 and pH 9.0–9.5 and specific activity was seen to be 0.013 μmoles of p-nitrophenyl phosphate/min/mg protein at 37 C at pH 4.5 and 0.00169 μmoles at pH 9.0. The ratio of acid to alkaline phosphatase was 7.7:1.0. The K_m for acid phosphatase (EC 3.1.3.2) was 0.5 mM with a V_max of 0.0128 units/mg protein and 0.2mM for alkaline phosphatase (EC 3.1.3.1) with a V_max of 0.00175 units/mg protein. Acid phosphatase activity was optimal at 60 C and alkaline at 37 C. Linearity of enzyme activity was observed with time after the first 15 min of incubation and with homogenate concentration. KCN at 20 mM inhibited 82% of activity at pH 9.0 but also 91.5% activity at pH 4.5. NaF at 10^?2M inhibited 92% of activity at pH 4.5 but had no effect at pH 9.0. The two flukicides rafoxanide and nitroxynil at 20mM had little effect on activity at pH 9.0 and pH 4.5. Enzyme activity at pH 4.5 was found to be greatest in the microsomal fraction with high activity in the lysosomal and soluble fractions. Histochemically, alkaline phosphatase was restricted to the excretory system, vitellaria, and uterus while acid phosphatase was found in the integument and gastrodermis.     

Moniliformis dubius: uptake of leucine and alanine by adults

Uptake of ¹⁴C-leucine by Moniliformis dubius in 2-min incubations was not linear with respect to substrate concentration and appeared to involve a combination of diffusion and mediated transport. During a 90-min incubation in 1 mM¹⁴C-leucine, the total pool of free leucine increased from an initial concentration of 0.46–12.21 mM; less than 2% of the absorbed leucine was metabolized during this time period. The concentrations of leucine in the pseudocoelomic fluid and extracts of body walls, measured before and after incubation, were the same in either case. Uptake of ¹⁴C-leucine was insensitive to the external concentration of Na⁺, Ca²⁺, Mg²⁺, K⁺, and Tris(hydroxymethyl) aminomethane.Uptake of ¹⁴C-alanine also appeared to be mediated, however, the alanine pool was not altered after a 30-min incubation in 1 mM¹⁴C-alanine. Following a 30-min incubation in ¹⁴C-alanine, only 38% of the absorbed radioactivity was present as labeled alanine; the remaining radioactivity was detected in aspartic acid, cysteic acid, taurine, and urea.     

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD⁺:isocitrate dehydrogenase and NADP⁺:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.     

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C₁₈ stationary phase and mobile phases of 20–40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4–5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the d- and l-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM β-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.     

Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

Urease fromAnabaena doliolum andAnacystic nidulans showed maximum activity at pH 7.0–7.4 at 40°C when measured in cell-free, phosphate-buffered extracts. It is a soluble enzyme located in cytoplasm. The apparent K_m forA. doliolum urease was 120 M. Anacystis nidulans urease exhibited biphasic kinetics (K_m=250 M and 1.66 mM). Enzyme, fully expressed in cells grown with urea, nitrate, or N₂, was repressed in ammonia-grown cells, but ammonia did not inhibit the activity in vitro. Incubation of algal cells in N₂ medium with chloramphenicol for 12 h caused degradation of urease. Cu²⁺ at 1 M inhibited the enzyme activity by 50%, whereas Co²⁺ and Ni²⁺ up to 20 M had no effect.p-Hydroxymercuribenzoate appeared to be a more powerful inhibitor of urease than acetohydroxamic acid.Address reprint requests to: c/o Prof. Robert Tabita, Department of Microbiology, Experimental Science Building #319, The University of Texas at Austin, Austin, TX 78712, USA.     

Effects of water gradients on soil enzyme activity and active organic carbon composition under Carex lasiocarpa marsh

The responses of soil enzyme activity of freshwater marsh, microbial biomass carbon (MBC), dissolved organic carbon (DOC) and aboveground biomass to water gradients were studied with Carex lasiocarpa pot culture experiment. The relationships between soil enzyme activity and MBC, DOC and aboveground biomass were discussed. The water gradients were W1, 15 cm; W2, ?5 cm; W3, ?5–5 cm; W4, submerged. The results indicated that acid phosphatase, invertase and urease activities were decreased with the increase of water level, while catalase activity was increased with moisture content increasing. Drying-wetting alternation (W3) increased soil enzyme activities if compared with W1. MBC content followed the order of W3 > W1 > W2 > W4, and the activities of invertase, urease and catalase were significantly positively correlated with MBC (p < 0.05). DOC content presented the order of W4 > W1 > W3 > W2, and the activities of urease and acid phosphatase were most significantly negatively correlated with DOC (p < 0.01). In addition, drying-wetting alternation promoted the growth of Carex lasiocarpa. When water submerged plants, the growth of Carex lasiocarpa was significantly inhibited. The aboveground biomass was positively related to soil enzyme activities. There were close relationships between the activities of invertase, urease and catalase and the growth situation of Carex lasiocarpa.     

The synthesis and evaluation of phenoxyacylhydroxamic acids as potential agents for Helicobacter pylori infections

Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC₅₀?=?0.061?±?0.003?μM) and intact cell (IC₅₀?=?0.89?±?0.05?μM), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds.