hymecromone and 4-methylumbelliferyl-caprylate

Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established.. A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies.. We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates.. The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

Topics: Biosensing Techniques; Enzyme Activation; Esterases; High-Throughput Screening Assays; Hydrolysis; Hymecromone; Models, Chemical; Polysorbates; Risk Assessment; Spectrometry, Fluorescence; Substrate Specificity

To detect Salmonella more efficiently and isolate strains more easily, a novel and simple detection method that uses an enrichment assay and two chromogenic reactions on a chromatography membrane was developed. Grade 3 chromatography paper is used as functionalized solid phase support (SPS), which contains specially optimized medium. One reaction for screening is based on the sulfate-reducing capacity of Salmonella. Hydrogen sulfide (H2S) generated by Salmonella reacts with ammonium ferric citrate to produce black colored ferrous sulfide. Another reaction is based on Salmonella C8 esterase that is unique for Enterobacteriaceae except Serratia and interacts with 4-methylumbelliferyl caprylate (MUCAP) to produce fluorescent umbelliferone, which is visible under ultraviolet light. A very low detection limit (10(1) CFU ml(-1)) for Salmonella was achieved on the background of 10(5) CFU ml(-1) Escherichia coli. More importantly, testing with more than 1,000 anal samples indicated that our method has a high positive detection rate and is relatively low cost, compared with the traditional culture-based method. It took only 1 day for the preliminary screening and 2 days to efficiently isolate the Salmonella cells, indicating that the new assay is specific, rapid, and simple for Salmonella detection. In contrast to the traditional culture-based method, this method can be easily used to screen and isolate targeted strains with the naked eye. The results of quantitative and comparative experiments showed that the visual detection technique is an efficient alternative method for the screening of Salmonella spp. in many applications of large-sized samples related to public health surveillance.

Topics: Bacteriological Techniques; Chromatography, Paper; Humans; Hydrogen Sulfide; Hymecromone; Salmonella; Salmonella Infections; Sensitivity and Specificity

Diacylglycerol lipase α (DAGLα) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLα as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLα activity. Two previously reported DAGLα surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLα activity in live cell assays using a human cell line stably expressing the human DAGLα transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLα assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Topics: Butyrates; Cell Line; Cell Survival; Drug Evaluation, Preclinical; Enzyme Assays; Enzyme Inhibitors; Halogenation; Humans; Hymecromone; Lipoprotein Lipase

The kinetics of chemical hydrolysis including neutral, acid- and base-catalyzed hydrolysis of 4-methylumbelliferyl caprylate (MUCAP) salmonella reagent were studied at different temperatures. The rate constants and activation parameters were determined by following the build-up of fluorescence peak of the hydrolysis product 4-methylumbelliferone (4-MU). The time scale of esterase enzyme hydrolysis caused by salmonella was compared with chemical hydrolysis as a background process.

Topics: Bacterial Typing Techniques; Hydrolysis; Hymecromone; Kinetics; Salmonella; Salmonella Infections

To study the sensitivity, specification, detection limit and the practical use of MUCAP method for the detection of Salmonella in water.. A comprehensive method of pre-enrichment + selective cultivation + initial screening with MUCAP + oxidase test + serological test was applied to check Salmonella in water samples spiked with bacteria and environmental water samples.. the sensitivity of MUCAP method was 100%, the specification was 80.8%, and the detection limit was 1 cfu/100 ml.. MUCAP method can effectively identify Salmonella in water and the testing time was shortening from 72h to 48h.

Topics: Bacteriological Techniques; Hymecromone; Indicators and Reagents; Salmonella; Sensitivity and Specificity; Water Microbiology

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Escherichia coli; Esterases; Fluorescent Dyes; Hydrolysis; Hymecromone; Molecular Sequence Data; Salmonella typhimurium; Sequence Alignment; Sequence Homology, Amino Acid

The 4-methylumbelliferyl-caprylate (MUCAP) was applied for the identification of Salmonella, it's specification, sensitity, and availability were reported. 65 strains of standard Salmonella, 48 strains of Salmonella isolated from foods growing on plates of HE, DHL, SS and MaConkey agar have been tested with MUCAP. All of them were identified as MUCAP positive; whereas, among the non-Salmonella tested only Pseudamonas spp., Aeromonas hydrophilia and Plesiomonas shigelloides were shown as positive, but they could be differentiated easily with Salmonella by means of oxidase test. The unspecific reaction of Serratia marcescens could be eliminated by using the plate medium which has been added of 1% sucrose. Both the sensitivity and specificity of the method were up to 97% more, and the operating of the method was very facility and to be done within few minutes.

Topics: Hymecromone; Indicators and Reagents; Meat; Salmonella; Sensitivity and Specificity

The utility of Rambach agar to identify Salmonella spp. was examined relative to its usefulness in clinical microbiology. Forty-four of 54 (82%) salmonella organisms isolated from faecal cultures and 66 of 82 (84%) salmonella stock cultures produced bright red colour colonies after 24 h incubation at 37 degrees C, whereas 48 of 54 (89%) salmonellae isolated from faecal cultures, and 74 of 82 (90%) salmonella stock cultures, yielded the bright red colour when the incubation time was extended to 48 h. Apart from Salmonella typhi and Salmonella paratyphi A the sensitivity of Rambach agar to detect salmonella strains belonging to five serogroups was 83% and 92% after 24 and 48 h of incubation, respectively. In contrast, other members of the family Enterobacteriaceae tested failed to give the bright red colour, except for one strain of Pseudomonas aeruginosa and another of Acinetobacter baumannii. The non-salmonella strains either gave a different colour--blue, green or orange--or were colourless. To supplement the use of Rambach agar in the detection of Salm. typhi and Salm. paratyphi A and other late or negative acid-producing salmonella species on this medium, the 4-methylumbelliferyl caprylate fluorescence (MUCAP) test was carried out, and this showed positive results with all the salmonella strains tested. These results suggest that while Rambach agar can not pre-identify Salm. typhi and Salm. paratyphi A, the use of a simple and rapid (MUCAP) test in combination would make it very useful to identify all Salmonella spp. after 24 h incubation.

Topics: Agar; Bacterial Typing Techniques; Fluorescence; Humans; Hymecromone; Pigmentation; Propylene Glycols; Salmonella

We have tested 750 Salmonella strains and 130 strains of other species of the family Enterbacteriaceae with the 4-methylumbelliferyl caprilate reagent (MUCAP) test. The MUCAP test is a fluorescence test for rapid identification of Salmonella strains. The non-Salmonella strains were strains sent for identification as suspected Salmonella strains and thus have phenotypes similar to those of Salmonella strains. All 748 tested Salmonella strains of subgroups I, II, III, and IV were positive in the MUCAP test. Of the two tested rare Salmonella subgroup V strains, one was positive and the other was negative. In the selected material containing strains with phenotypes similar to those of Salmonella strains, only one Hafnia alvei strain of 130 Enterbacteriaceae bacteria tested was positive. The fluorescence of the H. alvei strain, the six tested Salmonella dublin strains, and the positive Salmonella subgroup V strain was weaker than that of the other salmonellae. The MUCAP assay is simple and is performed within 5 min. With an almost 100% sensitivity for Salmonella strains, apart from a single Salmonella subgroup V strain, we found the MUCAP test to be a convenient complement to traditional biochemical identification methods, especially for atypical and unusual Salmonella strains.

Topics: Bacteriological Techniques; Enterobacteriaceae; Evaluation Studies as Topic; Fluorescent Dyes; Humans; Hymecromone; Salmonella; Serotyping