Faecal leukocyte esterase activity is an alternative biomarker in inflammatory bowel disease

Background: Leukocyte cytosolic proteins (e.g., calprotectin) are emerging biomarkers for inflammatory bowel disease. Leukocyte aryl esterase activity has been commonly used for sensitive detection of leukocytes in human body fluids such as urine. Urine test strip results are generally reported in categories. As automated strip readers allow quantitative data to be reported, sensitive quantitative detection of leukocytes in body fluids has become possible. Here, we explored the use of leukocyte esterase as a potential alternative faecal biomarker for inflammatory bowel disease. Methods: We evaluated leukocyte esterase activity in faecal extracts and compared Cobas u 411 (Roche) quantitative reflectance data with calprotectin concentration for 107 routine samples. Stability of leukocyte esterase for trypsin digestion was carried out by adding trypsin to the extract. Incubation occurred at 37 ° C for 24 h or 48 h. Results: Reproducibility of the reflectance signal was good (within-run imprecision: 6.1%; between-run imprecision: 6.2%). Results were linear in the range 10 3 – 10 6 WBC/100 mg faeces. The lower limit of detection was 4 WBC/ μ L and the lower limit of quantification was 5 WBC/ μ L. Stability of LE activity in stool and faecal matrix was good. An adequate correlation was obtained between leukocyte esterase activity and the faecal calprotectin concentration: log(y)  =  4.28 + 0.29log(x). In vitro experiments monitored the digestion of leukocyte esterase and faecal calprotectin. Leukocyte esterase activity was significantly less affected by trypsin activity than calprotectin immunoreactivity. Conclusions: Quantitative leukocyte esterase activity of faecal extracts provides information about the leukocyte count in the gut lumen. Leukocyte esterase is a promising and affordable alternative biomarker for monitoring inflammatory bowel disease

Studies on the non-specific esterases of Saccharomyces cerevisiae : a thesis in partial fulfillment [sic.] of the requirements for the degree of Master of Science in Microbiology at Massey University

Twenty wine-making and three laboratory strains of Saccharomyces cerevisiae were examined for non-specific esterases by Polyacrylamide Gel Electrophoresis. All wine-making strains contained the fast alleles of the Est 1 and Est 2 loci, confirming there is a selective advantage for the Est 1f and Est 2f genes in these strains. Only one wine-making strain carried the Est 3 and Est 4 genes, which was a much lower frequency than that published. The three laboratory strains all contained the Est 1f and Est 2s genes. A new non-specific esterase band, labelled Est 5, was identified by using a modified staining technique, which was apparently of low molecular weight as it travelled with the tracking dye front. Fast and slow alleles of Est 1 and Est 2 were determined to be charge allozymes. Est 2 proteins were considered to be polymeric, probably dimeric, and the Est 1 proteins to undergo post-translational modification. Difficulty in resolving the Est 4 band was overcome by adding Triton X-100 to cell suspensions before disruption, indicating this esterase protein may be particulate bound. Molecular weights were determined by Ferguson Plots to be 51,000 ± 10,000 daltons (Est 2), 60.000 ± 12,000 daltons (Est 3), 73,000 ± 15,000 daltons (Est l), and 113,000 ± 23,000 daltons (Est 4). No isolates of S. cerevisiae for comparison of allele frequencies could be made from mature locally-grown grapes, indicating that this species is rare in the New Zealand environment, which is in accordance with published studies. No "inducible" non-specific esterases were found in strains examined at different stages in the life cycle, or by growth in different media. The level of esterase activity in cells increased throughout aerobic growth in liquid media, but was quickly lost during fermentation. Esterase activity during sporulation also decreased. A non-specific esterase mutant was induced by ethyl methane-sulfonate and detected by the hydrolysis of α-naphthyl acetate incorporated into solid medium. This mutant lost expression of both Est If and Est 2s , as did subsequent mutants produced by hybridisation. Segregation of esterase-deficient to esterase-proficient spores after hybridisation, showed that two unlinked loci were involved in esterase suppression, both genes being unlinked to ade 1, Est 1 and mating type locus MAT. It is hypothesised these genes are a suppressor (SUP) and a mutated regulator (Reg Est− ). Gas Liquid Chromatography was used to quantitatively determine volatile ester concentrations produced during fermentation. Selected wine-making strains and diploid strains produced by micromanipulation and having different non-specific esterase compositions were fermented to the limit of their ethanol tolerance in Reisling Sylvaner grape juice and Complete Defined Medium. Ethyl acetate, ethyl propanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, 2-phenethyl acetate, n-hexyl acetate and iso-pentyl acetate were all quantitated. A maximum error of ±30% was determined for differences in ester concentration between two fermentations using the same strain. Correction for differences in fermentation ability by different strains was attempted, and the resulting ester concentrations compared qualitatively. Results indicate that differences in volatile ester concentrations between strains are not due to the esterase composition. The non-specific esterases probably have little if any influence on wine bouquet as the majority of ester production is late in fermentation when esterase activity has ceased

Effect of lithium on acetylcholine esterase activity, and isozyme pattern in developing chick brain

Acetylcholine Esterase is an enzyme, which hydrolyses acetylcholine and is used as a marker for cholinergic neural function. It is known to be involved in synaptogenesis. While on one hand it is known to be a marker for the developing chick brain it is also implicated in neurodegenerative diseases. In vertebrates the protein is synthesized by a single gene and undergoes alternative splicing to give 6-8 isoforms. Isozyme patterns of acetylcholine esterase have been suggested to be useful prognostic markers of neuronal degeneration. Lithium a well-known teratogen is known to induce apoptosis in the developing chick brain. Understanding the dynamics of acetylcholine esterase isoform pattern in lithium induced neural tissue damage would help elucidating the role of these isoforms in frank neurodegenerative diseases. We have therefore studied activity and isozyme pattern of acetylcholine esterase in lithium treated and control 7 day old developing chick brain and report the same

Regulation of the juvenile hormone titre in the Colorado potato beetle

Three main topics were investigated in regulation of the titre of juvenile hormone in haemolymph of the Colorado potato beetle ( Leptinotarsa decemlineata Say): enzymic breakdown of the hormone; binding and protection of the hormone by carrier proteins; the synthetic capacity of the corpora allata.Juvenile hormone was broken down by two major pathways: ester hydrolysis by esterases and hydration of the epoxide group by epoxide hydratases in tissue. In haemolymph of the beetle, juvenile hormone is solely broken down by juvenile hormone esterases. An in vitro method was developed to measure the catalytic activity of juvenile hormone esterase from haemolymph. High activities were observed in fourth-instar larvae and in beetles just before diapause. Lower activities were found in third- instar larvae and in beetles reared with long days, at diapause and after diapause. The juvenile hormone esterase was insensitive to diisopropylfluorophosphate (DFP), an inhibitor used to distinguish between carboxylesterases and esterases specific to juvenile hormone. Electrophoresis of the esterase from haemolymph showed one or more esterases specific to juvenile hormone.The short half-life of juvenile hormone measured in vivo and in vitro in the haemolymph and inhibition studies with Triton X-100 suggests that juvenile hormone esterases in haemolymph govern breakdown. Activities of juvenile hormone esterase correlate well with the juvenile hormone titre.The sharp changes in juvenile hormone esterase suggest that esterase activity is regulated. The mechanism was studied by supplying juvenile hormone and by microsurgery. Treatment of diapausing beetles with juvenile hormone itself or analogues caused an increase in activity of juvenile hormone esterase within 24 h. Ligation or removal of corpora allata suggested that this induction was an indirect effect of juvenile hormone. Transfer from short day to long day and treatment with hormone of beetles reared with short days prevented high activity of juvenile hormone esterase. Removal of corpora allata at emergence from beetles reared with short days resulted in the same. In beetles reared with short days the titre of hormone during the first days after adult emergence probably induces the rise in esterase. Esterase activity is thus most likely controlled indirectly by the hormone, via a centre in the brains (hormostate). The level of esterase activity is probably dependent on the sensitivity of this hormostate and on the titre of the juvenile hormone.In several insects juvenile hormone is transported bound to carrier proteins. In haemolymph of larval and adult Colorado potato beetles lipoproteins of high molecular weight (>100,00 daltons) were found, capable of binding juvenile hormone, its analogues, and palmitic acid. The lipoproteins were partially separated by gel permeation chromatography and electrophoresis on polyacrylamide gel. The binding characteristics of the lipoproteins indicate low affinity (K d ≈10 -5 M), low specificity and high binding capacity. The juvenile hormone complexed to lipoproteins was protected against esterases from haemolymph to some extent. Thus these carrier lipoproteins probably play little role in the regulation of the titre of juvenile hormone.In the last part of our investigations the activity of the corpora allata was measured in vitro. High activities were observed in beetles reared with long days and in beetles after emergence. In beetles reared with short days, amounts of hormone produced were intermediate until Day 6 after emergence, thereafter declining to a low value. During diapause, production remained low. The production by corpus allatum and the activity of juvenile hormone esterase were in good agreement with the titre of juvenile hormone. The corpora allata are probably the primary regulator of the hormone titre in the Colorado potato beetle.<p/

Cloned mouse cells with natural killer function and cloned suppressor T cells express ultrastructural and biochemical features not shared by cloned inducer T cells.

We have examined the morphology, cytochemistry, and biochemistry of mouse leukocyte subsets by analyzing cloned leukocyte populations specialized to perform different immunologic functions. Cloned cells expressing high-affinity plasma membrane receptors for IgE and mediating natural killer (NK) lysis and cloned antigen-specific suppressor T cells contained prominent osmiophilic cytoplasmic granules similar by ultrastructure to those of mouse basophils. Both clones also incorporated 35SO4 into granule-associated sulfated glycosaminoglycans, expressed a characteristic ultrastructural pattern of nonspecific esterase activity, incorporated exogenous [3H]5-hydroxytryptamine, and contained cytoplasmic deposits of particulate glycogen. By contrast, cloned inducer T cells lacked cytoplasmic granules and glycogen, incorporated neither 35SO4 nor [3H]5-hydroxytryptamine, and differed from the other clones in pattern of nonspecific esterase activity. These findings establish that certain cloned cells with NK activity and cloned suppressor T cells express morphologic and biochemical characteristics heretofore associated with basophilic granulocytes. However, these clones differ in surface glycoprotein expression and immunologic function, and the full extent of the similarities and differences among these populations and basophils remains to be determined

The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin

Biochemical studies on malathion resistance, inheritance and association of carboxylesterase activity in brown planthopper, Nilaparvata lugens complex in Peninsular Malaysia.

Two sympatric populations of brown planthopper (BPH), one from rice and the other from Leersia hexandra were collected from each of five locations in Malaysia. All the tested malathion-resistant individuals of the rice BPH population and F1 generation (cross between malathion-resistant [usually caught on rice] and malathion-susceptible [usually caught on Leersia]) showed high esterase activity, while all malathion-susceptible individuals on L. hexandra showed low esterase activity. In the F2 generation,all the individuals tested against malathion were approximately 75% resistant and 25% susceptible and the inheritance pattern of esterase activity (high and low esterase activity) segregated in the same manner to a 3: 1 ratio. This confirms that resistance to malathion is mono-factorial and inheritance pattern of esterase activity is also linked to malathion resistance. Carboxylesterase or total esterase activity in BPH is inherited in a simple Mendelian fashion that is encoded by a single dominant gene. For the total esterase assay, average esterase activity levels in the rice-infesting population ranged from 17.64 to 19.37 nmoles1-napthol/mg protein while that in the Leersia-infesting population ranged from 5.29 to 6.11 nmoles 1-napthol/mg protein. In terms of esterase activity, the two sympatric Ni-laparvata lugens populations separated into two distinct groups. Results based on the tube color intensity test showed 96% and 98% resistant and susceptible individuals were present in the rice- and Leersia-infesting populations, respectively. In a filter paper test, the rice-infesting population had 94% with high esterase activity while the Leersia-infesting population had 96% with low esterase activity

Characterization of esterase activity from the bacteria, Francisella tularensis, the causative agent of tularemia

Francisella tularensis is the bacteria responsible for causing the disease tularemia and is listed as one of the top three-biowarfare agents. Among the proteins essential to the virulence and infectivity of F.tularensis are multiple esterases, which are enzymes that break down various ester, thioester, and amide bonds. In this project, the catalytic activity, substrate speci fi city, and structure of a putative esterase from F.tularensis was studied. Latent fluorophores based on the molecule, fluorescein, were unmasked by the enzymatic activity of the esterase and the increase in fluorescence was measured over time to determine how well the e tcrase recognized different substrates. The esterase FlT258 from F. IIJlarensis activated a variety of simple latent fluorophore substrates with catalytic efficiencies ranging from 5075 M s for a simple propyl ester to 294.8 M\u27 \u27s for a teniary e ter. These simple substrates were recognized by the esterase with KM va lues ranging from 0.54 to 2 1.4 f,M , and sterically occluded substrates had significantly reduced kinetic turnover (kc ,) compared to the simplest substrates. In addition to the wild type esterase, the kinetic a tivity of five different variants of the esterase with single amino acid mutations were characterized against two latent fluorophore substrates to determine more information about the binding pocket of the esterase. The kinetic activity of each of the variants decreased significantly from the wild-type enzyme activity and indicated that the binding pocket is fairly invariant to substitution. Activity, 3D structure, and primary structure comparisons suggest that this esterase belongs to the carboxylesterasc family. Although lillie is known about the specific biological role of FTI 0258C and other carboxylesterases from its fanlily, the promiscuity of its enzymatic eclJ\ll) ould Ix uoollO dc\ I P f\u27OlmlJ:tl Jru m,\u3edcl thaI ulIlll 1M Un

Esterase Activity from the Germinated Jatropha curcas Seeds in Different Extraction buffers.

The buffer solution plays a major role in protein stability and activity, thereby making the selection of a buffer to achievemaximum activity for a protein will be a formidable challenge. The present work constitutes an extension of this investigation to esterases from germinated Jatrophacurcas seeds. The 0.1 M NaCl solution, 0.1 M phosphate buffer pH7.0, 0.1 M citrate buffer pH 5.0 and 0.1 M Tris-HCl buffer pH 8.5, 0.1 M NaOH and distill water were used to extract protein from germinated Jatrophacurcas seeds. The esterase activity and specific activity for NaCl solution, phosphatebuffer, citrate buffer, Tris-HCl buffer, NaOH and Distilled water was 9.07, 8.6, 8.2, 6.46, 0.07 and 4.98 μmoles/min/gm and 0.09258, 0.0905, 0.088, 0.0715 0.0003 and 0.081 IU/mg, respectively.The Native-PAGE analysis showed the esterase enzyme activity in different extraction buffer.Among 13 esterase bands, 8 esterolytic bands were major bands (band no 1,3, 6, 7, 8, 11, 12 and 13)and remaining were minor bands.The amount of proteins and esterase activity were found to bethe highest when extracted with 0.1 M NaCl solution

Esterase activity and isoenzymes in relation to morphogenesis in Mammillaria gracillis Pfeiff. tissue culture

Cactus Mammillaria gracillis Pfeiff. (Cactaceae), cultivated in vitro, spontaneously switches from an organised to unorganised way of growth, producing a habituated organogenic callus which regenerates normal and hyperhydric shoots without the addition of any growth regulators. Tumour tissues, induced by A. tumefaciens wild strain B6S3 (tumour TW) and rooty mutant GV3101 (tumour TR), do not express any organogenic potential. The esterase (arylesterase EC 3.1.1.2 and carboxylesterase EC 3.1.1.1) activity and isoenzyme pattern of morphologically different cactus tissues: shoot, callus, hyperhydric regenerant, tumours TW and TR, were compared. Tissue samples were frozen at –80 °C and lyophilized before protein extraction. Two esterase substrates, 1- and 2-naphthylacetate, were used. Esterase activity of all tissues varied during the period of one subculture. In shoots and tumours, the highest esterase activity for both substrates was measured on the 21st day, while in the callus and hyperhydric regenerants the highest activity was on the 7th day. Esterases were separated electrophoretically in polyacrylamide gradient gels under non-denaturating conditions. In total, 13 isoesterases, reacting with both substrates, were resolved. No differences in isoenzyme profile were noticed in correlation with the age of tissues, but the esterase activity varied among tissues. The significance of these results is discussed