A Broad Spectrum Racemase in \u3cem\u3ePseudomonas putida\u3c/em\u3e KT2440 Plays a Key Role in Amino Acid Catabolism

The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the L- and D-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of D-Lys in the growth medium. An alr gene knockout strain (Δalr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of L-Lys and L-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of L-Lys by virtue of its ability to convert L-Lys to D-Lys, which is further catabolized through the L-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of L-Arg, although the pathway by which D-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism

Amino Acid Racemase Enzyme Assays

Amino acid racemases are enzymes that invert the α-carbon stereochemistry of amino acids (AAs), interconverting amino acids between their L- and D-enantiomers in a reversible reaction. In bacteria, they are known to have catabolic physiological functions but are also involved in the synthesis of many D-AAs, including D-glutamate and D-alanine, which are necessary components of the peptidoglycan layer of the bacterial cell wall. As such, amino acid racemases represent significant targets for the development of bactericidal compounds. Amino acid racemases are also regarded by the biotechnological industry as important catalysts for the production of economically relevant D-AAs. Here, we provide a detailed protocol using high performance liquid chromatography (HPLC) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, also Marfey’s reagent) for the characterization of novel amino acid racemases. The protocol described here was designed to obtain accurate kinetic parameters (kcat, KM values). Enzyme concentrations and reaction times were optimized so as to minimize the reverse reaction, which can confound results when measuring racemase reactions

A Broad Spectrum Racemase in \u3cem\u3ePseudomonas putida\u3c/em\u3e KT2440 Plays a Key Role in Amino Acid Catabolism

The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the L- and D-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of D-Lys in the growth medium. An alr gene knockout strain (Δalr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of L-Lys and L-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of L-Lys by virtue of its ability to convert L-Lys to D-Lys, which is further catabolized through the L-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of L-Arg, although the pathway by which D-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism

Clinical cases of pulmonary tuberculosis as a result of TNF antagonist therapy

През последните 12 години антагонистите на TNF са били успешно използвани за лечение на много пациенти страдащи от хронични възпалителни заболявания. Това лечение увеличава риска от туберкулоза до 25 пъти. Дължи се на факта, че TNF и TNF-рецепторите играят важна роля в медиирането на имунния отговор при остри и хронични възпаления. Ето защо всички пациенти, на които предстои подобно лечение трябва да бъдат подложени на стриктна оценка за изключване на активна и латентна туберкулозна инфекция. През 2010 г. беше публикуван консенсус на TBNET, озаглавен „Рискът от туберкулоза, свързан с лечение с TNF антагонисти`.В изложението са представени два случая на белодробна туберкулоза, които са наблюдавани в хода на провеждано лечение с TNF антагонисти при болни с доказани възпалителни заболявания на червата.During the past 12 years TNF antagonists have been successfully used for the treatment of many patients suffering from chronic inflammatory diseases. This treatment increases the risk of tuberculosis up to 25 times. This is due to the fact that TNF and TNF-receptors play an important role in mediating the immune response in acute and chronic inflammation. Therefore all patients undergoing such treatment should be subject to rigorous assessment to exclude active and latent tuberculosis infection. In 2010 a TBNET consensus was published entitled "The risk of tuberculosis related to TNF therapies." In this article we present two cases of pulmonary tuberculosis, which were observed in the course of an ongoing treatment with TNF antagonists in patients with proven inflammatory bowel disease

IMMUNOMODULATORS IN PULMOLOGIGAL PRACTICE

No abstrac

ANTISTREPTOLYSIN `0` TITER IN PATIENTS WITH VIRAL HEPATITIS

No abstract

AN ATTEMPT AT NOSOLOGICAL AND ETIOLOGICAL DIARRHEA SYNDROME DECODING IN PATIENTS WITH PRESUMED ENTERAL INFECTION

No abstrac

Building block libraries and structural considerations in the self-assembly of polyoxometalate and polyoxothiometalate systems

Inorganic metal-oxide clusters form a class of compounds that are unique in their topological and electronic versatility and are becoming increasingly more important in a variety of applications. Namely, Polyoxometalates (POMs) have shown an unmatched range of physical properties and the ability to form structures that can bridge several length scales. The formation of these molecular clusters is often ambiguous and is governed by self-assembly processes that limit our ability to rationally design such molecules. However, recent years have shown that by considering new building block principles the design and discovery of novel complex clusters is aiding our understanding of this process. Now with current progress in thiometalate chemistry, specifically polyoxothiometalates (POTM), the field of inorganic molecular clusters has further diversified allowing for the targeted development of molecules with specific functionality. This chapter discusses the main differences between POM and POTM systems and how this affects synthetic methodologies and reactivities. We will illustrate how careful structural considerations can lead to the generation of novel building blocks and further deepen our understanding of complex systems

Photoproduction of pi0 omega off protons for E(gamma) < 3 GeV

Differential and total cross-sections for photoproduction of gamma proton to proton pi0 omega and gamma proton to Delta+ omega were determined from measurements of the CB-ELSA experiment, performed at the electron accelerator ELSA in Bonn. The measurements covered the photon energy range from the production threshold up to 3GeV.Comment: 8 pages, 13 figure