Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF

Background To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme. Results Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala. Conclusion MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development

Altering Enzymatic Activity: Recruitment of Carboxypeptidase Activity into an RTEM β-Lactamase/Penicillin-Binding Protein 5 Chimera

The D-Ala-D-Ala carboxypeptidases/transpeptidases (penicillin-binding proteins, PBPs) share considerable structural homology with class A β-lactamases (EC 3.5.2.6), although these β-lactamases have no observable D-Ala-D-Ala carboxypeptidase activity. With the objective of recruiting such activity into a β-lactamase background, we have prepared a chimeric protein by inserting a 28-amino acid segment of PBP-5 of Escherichia coli in place of the corresponding region of the RTEM-1 β-lactamase. The segment thus inserted encompasses two residues conserved in both families: Ser-70, which forms the acyl-enzyme intermediate during β-lactam hydrolysis, and Lys-73, whose presence has been shown to be necessary for catalysis. This chimera involves changes of 18 residues and gives a protein that differs at 7% of the residues from the parent. Whereas RTEM β-lactamase has no D-Ala-D-Ala carboxypeptidase activity, that of the chimera is significant and is, in fact, about 1% the activity of PBP-5 on diacetyl-L-Lys-D-Ala-D-Ala; in terms of free energy of activation, the chimera stabilizes the transition state for the reaction to within about 2.7 kcal/mol of the stabilization achieved by PBP-5. Furthermore, the chimera catalyzes hydrolysis exclusively at the carboxyl-terminal amide bond which is the site of cleavage by D-Ala-D-Ala carboxypeptidase. Though containing all those residues that are conserved throughout class A β-Lactamases and are thought to be essential for β-lactamase activity, the chimera has considerably reduced activity ({approx} 10^-5) on penams such as penicillins and ampicillins as substrates. As a catalyst, the chimera shows an induction period of {approx} 30 min, reflecting a slow conformational rearrangement from an inactive precursor to the active enzyme

Answering the Calls of "What's Next" and "Library Workers Cannot Live by Love Alone" through Certification and Salary Research

Members and staff of the American Library Association (ALA) worked diligently over more than a decade to develop a certification program for public library managers. Spurred by a long-standing trend in many other terminal-degree professions that have post-degree, voluntary certifications, the Certified Public Library Administrator Program was born. Legal authority recommended the establishment of a service organization, a 501(c)(6) to manage the program, which has become one of several programs that will be offered to library employees under the imprimatur of ALA. After the American Library Association???Allied Professional Association (ALA-APA) was instituted, advocacy for salary improvement initiatives was appended to the mission. One means of salary advocacy was to improve available data by expanding the scope and usefulness of the ALA Survey of Librarian Salaries, which resulted in the ALA-APA Salary Survey: Non-MLS???Public and Academic, conducted in 2006 and 2007 to collect salary data from more than sixty positions in the field that do not require a master's degree in Library Science. The experience of establishing two certification programs, the Certified Public Library Administrator Program (CPLA??) and the Library Support Staff Certification Program, has been a study in creating new national models of professional development. This article will also discuss the insights that have emerged from fulfilling elements of ALA strategic plans concerning the needs of support staff through certification and the salary survey.published or submitted for publicatio

Closing the Loop: How an information literacy assessment plan contributes to first-year student success at UT Austin

Before attending the assessment track of Immersion I struggled with writing an assessment plan for my unit, which works collaboratively with faculty members to support information literacy in first-year seminar courses. Through Immersion, I had a breakthrough in my thinking about how to feasibly assess the mixture of teaching techniques and learning outcomes used throughout my unit to support the diversity of courses we work with. I brought the insights gained through work with my Immersion instructors and cohort members home, and my colleagues and I finished and enacted a comprehensive assessment plan. My poster will cover highlights of our plan, focusing on changes that we have made based on what we learned about student learning through assessment. I plan to feature: 1) How we are using individual classroom and course-level assessment methods as part of our program level assessment. 2) How we worked with faculty members to assess course-integrated information literacy in both large classes and small seminars. 3) How we have used the evidence we gathered to improve our program. 4) How the process of working together to create an assessment plan has increased both confidence in using student learning assessment methods and communication about assessment within my unit. I plan to interact with viewers by sharing what we have accomplished through assessment since my Immersion experience, and inviting them to discuss their experiences with assessment and successful methods they have used. This exchange of ideas will contribute to the ever-growing culture of assessment within our profession.UT Librarie

Probing the Reaction Mechanism of the D-ala-D-ala Dipeptidase, VanX, by Using Stopped-Flow Kinetic and Rapid-Freeze Quench EPR Studies on the Co(II)-Substituted Enzyme

In an effort to probe the reaction mechanism of VanX, the D-ala-D-ala dipeptidase required for high-level vancomycin resistance in bacteria, stopped-flow kinetic and rapid-freeze quench EPR studies were conducted on the Co(II)-substituted enzyme when reacted with d-ala-d-ala. The intensity of the Co(II) ligand field band at 550 nm decreased (ε550 = 140 to 18 M-1 cm-1) when VanX was reacted with substrate, suggesting that the coordination number of the metal increases from 5 to 6 upon substrate binding. The stopped-flow trace was fitted to a kinetic mechanism that suggests the presence of an intermediate whose breakdown is rate-limiting. Rapid-freeze quench EPR studies verified the presence of a reaction intermediate that exhibits an unusually low hyperfine constant (33 G), which suggests a bidentate coordination of the intermediate to the metal center. The EPR studies also identified a distinct enzyme product complex. The results were used to offer a detailed reaction mechanism for VanX that can be used to guide future inhibitor design efforts

Modulation of 5-Aminolevulinic acid mediated photodynamic therapy induced cell death in a human lung adenocarcinoma cell line

Photodynamic therapy (PDT) is a cancer treatment involving the administration of a photosensitising drug which selectively accumulates in tumor tissue, followed by irradiation with appropriate wavelength light. It triggers photochemical reactions inducing reactive oxygen species (ROS) production with the consequent cellular damage, which ultimately leads to cell death. Porphyrins are the only photosensitizers (PSs) endogenously synthesized by means of administration of the biological precursor, 5- aminolevulinic acid (ALA). Several antioxidants and ROS scavenger agents: reduced glutathione (GSH), mannitol (Man), l-tryptophan (Trp), ascorbate (Asc) and trolox (Trx), were assayed to determine their ability to modulate ALA-based PDT (ALA-PDT); it was performed on A549 human lung adenocarcinoma cells, by incubating with 1mM ALA for 3 hr and followed by irradiation with or without 1 hr pre-incubation with the modulators. They were previously tested for possible cytotoxicity/ photoactivity in concentrations ranging from 0.01 to 20 mM. The ratio between cell survival after ALA-PDT in the presence and in the absence of the scavenger agent (protection grade: PG) was determined, and the concentration showing no cytotoxicity/ photoactivity and providing the highest PG was used in the subsequent experiments. ALA-PDT alone induced a high percentage of apoptotic cell death (98.4 ± 3.5%) as revealed by acridine orange/ethidium bromide staining and AnnexinV-FITC/propidium iodide labelling. Pre-incubation with the modulators at their highest PG concentration significantly reduced apoptotic cells to 48.3 ± 2.7% (Asc), 58.8 ± 4.2 (Trx), 78.5 ± 3.1% (GSH), 64.3 ± 1.6% (Man), 74.6 ± 2.3% (Trp). ROS involvement in early cell death induction after ALA-PDT was tested by flow cytometry using the fluorescent probes dihydro-dichlorofluorescein diacetate (H2-DCFDA) and methoxyvinylpyrene (MVP) for detection of peroxides and singlet oxygen, respectively. ROS production increased after ALA-PDT (H2-DCFDA positive cells, control: 1.1 ± 0.1 %; 10 min-PDT: 69.3 ± 5.6%; MVP positive cells, control: 0.65 ± 0.35%; 10 min-PDT: 83.5 ± 1.9%). Asc prevented peroxide formation (H2-DCFDA positive cells: 50.7 ± 2.8%) and mostly prevented singlet oxygen increase (MVP positive cells: 25.4 ± 5.2%) whereas Trx limited peroxides formation (H2-DCFDA positive cells: 20.8 ± 0.5%), but did not significantly affected singlet oxygen production (MVP positive cells: 73.6 ± 3.4%). Selective scavenger mediated protection against PDT-induced cell death, and direct detection of specific pro-oxidative agents, entail the strong involvement of ROS in ALA-PDT-mediated tumor eradication, suggesting that undesired photodamage to normal tissue might be attenuated by administration of antioxidant agents.Fil: Teijo, Maria Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Diez, Berenice Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Battle, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Fukuda, Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Investigaciones sobre Porfirinas y Porfirias. Universidad de Buenos Aires. Centro de Investigaciones sobre Porfirinas y Porfirias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Photodynamic therapy of prostate cancer by means of 5-aminolevulinic acid-induced protoporphyrin IX - In vivo experiments on the dunning rat tumor model

Objective: In order to expand the use of photodynamic therapy (PDT) in the treatment of prostate carcinoma (PCA), the aim of this study was to evaluate PDT by means of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX ( PPIX) in an in vivo tumor model. Methods: The model used was the Dunning R3327 tumor. First of all, the pharmacokinetics and the localization of PPIX were obtained using fluorescence measurement techniques. Thereafter, PDT using 150 mg 5-ALA/kg b.w.i.v. was performed by homogenous irradiation of the photosensitized tumor (diode laser lambda = 633 nm). The tumors necrosis was determined histopathologically. Results: The kinetics of PPIX fluorescence revealed a maximum intensity in the tumor tissue within 3 and 4.5 h post-application of 5-ALA. At this time, specific PPIX fluorescence could be localized selectively in the tumor cells. The PDT-induced necrosis (n = 18) was determined to be 94 B 12% (range 60-100%), while the necrosis of the controls ( n = 12) differs significantly (p < 0.01), being less than 10%. Conclusion: These first in vivo results demonstrate the effective potential of 5-ALA-mediated PDT on PCA in an animal model. Copyright (C) 2004 S. Karger AG, Basel

Training entrepreneurial culture in enterprises of the Republic of Moldova

This article addresses the problems related to the formation of organizational culture in the Republic of Moldova. There are reviewed the results of research carried out in the business of producing and conclusions that are made. It highlighted the role of human resources training in organizational culture. The paper highlights the preconditions for the creation of an entrepreneurial culture.Organization culture; Training; Entrepreneurial culture