Quantification of ampicillin in bovine milk by coupled-column ultrahigh-performance liquid chromatography-tandem mass spectrometry.

This work reports the use of two-dimensional (2D) liquid chromatography system coupled with a tandem mass spectrometry for the quantification of ampicillin in bovine milk. A restrict access media column (RAM-BSA C(8) , 50 × 2.1 mm, Luna, 10 µm, 100 Å) was used in the first dimension in order to exclude macromolecules, while an ACQUITY UPLC BEH C(18) (50 × 2.1 mm, 1.7 µm) column was used in the second dimension. Three different channels of selected reaction monitoring (SRM) were used: 350 > 106 m/z, 350 > 160 m/z, and 350 > 192 m/z. The first transition was used for the quantification (higher intensity), and latter two for confirmation. The developed method is simple and requires a total analysis time of only 14 min/sample. The sample treatment involved only a centrifugation step for 20 min. The validated method has been successfully applied to monitor AMP residues in raw milk samples. To our knowledge, this is the first study to report the use of ultrahigh-performance liquid chromatography (UHPLC) in 2D configuration.

Acetylcholinesterases from Leaf-Cutting ant Atta sexdens: Purification, Characterization, and Capillary Reactors for On-Flow Assays.

Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs from Atta sexdens which were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively.

Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.

Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening.

Functional characterization of a yellow laccase from Leucoagaricus gongylophorus.

In this work we have identified, using mass spectrometry, two laccases produced by Leucoagaricus gongylophorus. One of them, Lac1Lg, was isolated, purified and characterized. Lac1Lg, a monomeric enzyme, was studied using ABTS and syringaldazine substrates. Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine. The interference of several metal ions and substances in the laccase activity were evaluated. Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases. Lac1Lg also was able to oxidize non-phenolic substrate (anthracene) in the absence of an exogenous mediator, showing that the enzyme has potential to explore in biotechnological processes. Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

Molecular and kinetic characterization of two extracellular Xylanases isolated from Leucoagaricus gongylophorus.

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km = 14.7 ± 7.6 mg mL(-1). Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km = 2.2 ± 0.5 mg mL(-1). XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km = 7.4 ± 2.0 mg mL(-1)) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.

Effect of water-bath post-polymerization on the mechanical properties, degree of conversion, and leaching of residual compounds of hard chairside reline resins.

OBJECTIVES: This study evaluated the effect of water-bath post-polymerization at 55 degrees C for 10 min (WB) on the content and leaching of residual compounds, degree of conversion, flexural strength, and hardness of hard chairside reline resins (Kooliner: K, New Truliner: N, Ufi Gel hard: U, and Tokuso Rebase Fast: T). METHODS: Leaching experiments were made by storing specimens (n=48) in artificial saliva at 37+/-1 degrees C and analyzing residual monomers and plasticizer by HPLC. Analysis of residual monomer and plasticizer content (n=48) was also made by HPLC. Degree of conversion (n=40) was analyzed by using FT-Raman spectroscopy. A 3-point loading test was used to evaluate the flexural strength of the specimens (n=80). One fragment of each flexural test specimen was then submitted to Vickers microhardness test. RESULTS: WB produced a significant decrease (p<0.050) in the amount of residual compounds eluted from the materials within the first hour of immersion. With the exception of material U, WB decreased the duration of release of the residual compounds evaluated. All materials evaluated exhibited significantly (p<0.050) lower values of residual monomer and plasticizer (material N) after WB compared with the control groups. WB increased the degree of conversion of K and T resins and the hardness of N, K, and T resins (p<0.050). Only material K showed an increase in flexural strength after WB (p<0.050). SIGNIFICANCE: Immersion of relined dentures in water at 55 degrees C for 10 min can be used to reduce the amounts and duration of release of residual compounds and improve mechanical properties of some of the materials evaluated.

Magnetic resonance imaging in the diagnosis of pelvic floor disorders.

Vaginal prolapse due to pelvic floor dysfunction occurs frequently in postmenopausal women. The disease usually involves all compartments of the vagina, so that isolated defects are uncommon. In advanced disease, it can be difficult to identify which organs are prolapsed, owing to the large bulge in vaginal area. Accurate diagnosis of pelvic floor defects, actual prolapsed organs, and presence of any coexisting abnormalities are essential to correctly plan surgical reconstruction and minimize the risk of recurrence. In this review, we discuss the existing imaging modalities available to evaluate pelvic prolapse, emphasizing the role of dynamic magnetic resonance imaging.