An Improved Spectrophotometric Phospholipase $A_2$ Assay Using 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphatidylcholine as Substrate and Lipoxygenase as Coupled Enzyme

Soccio, Mario,Trono, Daniela,Laus, Maura N.,Pastore, Donato 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.4

An improved spectrophotometric assay of phospholipase $A_2$ ($PLA_2$) activity based on the coupled $PLA_2$/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine ($PC_{LIN}$) as substrate is reported. The $PLA_2$-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 ($3{\mu}L/{\mu}mol$ phospholipid) as surfactant and of ethanol ($15{\mu}L/mL$ reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory $PLA_2$ from honey bee venom (HBV-$PLA_2$). The enzyme did not discriminate among $PC_{LIN}$, phosphatidylcholine, and phosphatidylethanolamine, but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine ($PC_{DILIN}$). Nevertheless, the use of $PC_{DILIN}$ is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine, are known to be oxidized by LOX. HBV-$PLA_2$ showed maximal activity at pH 9.0, hyperbolic kinetics ($K_m$, $74.2{\pm}2.9{\mu}M$; $V_{max}$, $827{\pm}7{\mu}mol/min/mg$ protein) and competitive inhibition ($K_i$ about $5{\mu}M$) by palmityl trifluoromethyl ketone, a classical $PLA_2$ inhibitor. Interestingly, the HBV-$PLA_2$/soybean LOX-1 coupled reactions also allow an accurate assay of $PC_{LIN}$ concentration. In the whole, these results demonstrate that this improved $PLA_2$/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.

Detection of Streptococcus dysgalactiae subsp. equisimilis in equine nasopharyngeal swabs by PCR

Silvia Preziuso,Fulvio Laus,Aurora Romero Tejeda,Carlo Valente,Vincenzo Cuteri 대한수의학회 2010 JOURNAL OF VETERINARY SCIENCE Vol.11 No.1

Streptococcus (S.) dysgalactiae subsp. equisimilis is responsible for severe diseases in humans, including primary bacteraemia, pneumonia, endocarditis, and toxic shock syndrome. Infection in some animal species can also occur, although a few studies have looked into cross-species infectivity. In horses, S. equisimilis is generally considered infrequent or opportunistic, but has recently been isolated from cases of strangles-like disease. Rapid and sensitive diagnostic techniques could enable epidemiological studies and effective investigation of outbreaks involving these bacteria. In this study, PCR protocols previously described in cattle and in humans to detect the species S. dysgalactiae and the subspecies equisimilis were evaluated to detect specific sequences in equine samples. For this purpose, 99 monolateral nasal swabs were collected from horses from stud farms with a history of S. equisimilis infection and were tested blindly by bacteriological isolation and by single and duplex PCR. DNA for PCR was extracted both from the colonies grown on agar media and from enrichment broth aliquots after incubation with nasal swab samples. S. equisimilis was identified by bacteriological isolation in 23 out of 99 swab samples, and PCR assays on these colonies were fully concordant with bacteriological identification (kappa statistic = 1.00). In addition, PCR of the enrichment broth aliquots confirmed the bacteriological results and detected S. equisimilis in 6 samples more than the bacteriological examination (kappa statistic = 0.84). The PCR protocols appeared to be reliable for the rapid identification of S. equisimilis in equine nasal swab samples, and could be useful for microbiological diagnosis.

Contributed Mini Review : The uniqueness of the plant mitochondrial potassium channel

Donato Pastore,Mario Soccio,Maura Nicoletta Laus,Daniela Trono 생화학분자생물학회(구 한국생화학분자생물학회) 2013 BMB Reports Vol.46 No.8

An Improved Spectrophotometric Phospholipase A2 Assay Using 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3- Phosphatidylcholine as Substrate and Lipoxygenase as Coupled Enzyme

Mario Soccio,Donato Pastore,Daniela Trono,Maura N. Laus 한국응용생명화학회 2013 Applied Biological Chemistry (Appl Biol Chem) Vol.56 No.4

An improved spectrophotometric assay of phospholipase A2 (PLA2) activity based on the coupled PLA2/lipoxygenase (LOX) reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PCLIN) as substrate is reported. The PLA2-mediated release of free linoleate is continuously monitored by following the absorbance increase at 234 nm caused by its conversion into the conjugated diene hydroperoxide catalyzed by the coupled soybean LOX-1 reaction. The new protocol includes the use of Tween 20 (3 μL/μmol phospholipid) as surfactant and of ethanol (15 μL/mL reaction mixture), that ensure clearness of reaction mixture and linear increase of absorbance in the course of reaction. This method was tested on a purified secretory PLA2from honey bee venom (HBV-PLA2). The enzyme did not discriminate among PCLIN, phosphatidylcholine, and phosphatidylethanolamine,but showed the highest rate using 1,2-dilinoleoyl-sn-glycero-3-phosphatidylcholine (PCDILIN). Nevertheless,the use of PCDILIN is not recommended, as it may induce an overestimation of enzyme activity, because not only the free linoleate, but also the reaction product 1-linoleoyl-lysophosphatidylcholine,are known to be oxidized by LOX. HBV-PLA2showed maximal activity at pH 9.0, hyperbolic kinetics (Km,74.2±2.9 μM; Vmax, 827±7 μmol/min/mg protein) and competitive inhibition (Ki about 5 μM) by palmityl trifluoromethyl ketone, a classical PLA2 inhibitor. Interestingly, the HBV-PLA2/soybean LOX-1 coupled reactions also allow an accurate assay of PCLIN concentration. In the whole, these results demonstrate that this improved PLA2/LOX assay allows a very accurate, simple, and rapid measurement of enzyme activity and substrate concentration.

School and Community Partnerships in Emergent Democracies: Narratives and Evidence from the Philippines

Rohaiba B. Radiamoda,Hsueh-Hua Chuang,Ronald A. Pernia,Magnolia A. Laus 부경대학교 글로벌지역학연구소 2023 Journal of Global and Area Studies(JGA) Vol.7 No.2

School and community partnerships are considered effective for achieving educational reforms, yet this type of collaborative enterprise continues to suffer from a crisis of relevance and sustainability up to the present. Hence, this study aims to identify factors that hinder the actualization of school–community partnerships. Using a case study approach, we conducted key-informant interviews, focus group discussions, and field observations in a public school in Cebu, Philippines. Through a thematic analysis of the data and anchored on Epstein’s Theory of Overlapping Influence (OSI), the study found that school leadership, sense of reciprocity, dynamics of local politics, and parent’s self-efficacy are the key factors that affect the practice of effective school and community partnerships. We also highlight that distinctive school contexts dictate how such partnerships are organized. One interesting finding indicates that the “diffusion of responsibility” may be detrimental. With the implementation of school-based management, the subjective diffusion of responsibility to “many leaders” results to finger-pointing and less proactive behavior. The spirit of school and community partnerships is in proper communication and effective collaboration―a critical dimension of the OSI model; without it, its purest intentions are most likely to fail. In an ironic twist of fate, decentralization of education, which is supposed to decenter the responsibilities of localities and peripheries, may actually hurt (instead of help) substantive educational and public policies in emergent democracies.