Factors Defining the Functional Oligomeric State of Escherichia coli DegP Protease

Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP

Guidelines of the Polish Respiratory Society for diagnosis and treatment of idiopathic pulmonary fibrosis

Introduction: This document presents the guidelines of the Polish Respiratory Society (PTChP, Polskie Towarzystwo Chorób Płuc) for diagnosis and treatment of idiopathic pulmonary fibrosis (IPF), developed by a group of Polish experts.Material and methods: The recommendations were developed in the form of answers to previously formulated questions concer-ning everyday diagnostic and therapeutic challenges. They were developed based on a current literature review using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology.Results: We formulated 28 recommendations for diagnosis (8), pharmacological treatment (12) as well as non-pharma-cological and palliative therapy (8). The experts suggest that surgical lung biopsy (SLB) not be performed in patients with the probable usual interstitial pneumonia (UIP) pattern, with an appropriate clinical context and unanimous opinion of a  multidisciplinary team. The experts recommend using antifibrotic agents in IPF patients and suggest their use irrespective of the degree of functional impairment. As regards non-pharmacological and palliative treatment, strong re-commendations were formulated regarding pulmonary rehabilitation, oxygen therapy (in patients with chronic respiratory failure), preventive vaccinations as well as referring IPF patients to transplant centres. Table 1 presents an aggregate list of recommendations.Conclusions: The Polish Respiratory Society Working Group developed guidelines for IPF diagnosis and treatment

Characteristic of energy-reduced functional foods

Zgodnie z Rozporządzeniem (WE) Nr 1924/2006 określenie „o obniżonej zawartości”, również kaloryczności, może być stosowane w stosunku do produktów, w których obniżenie zawartości danego składnika wyniosło co najmniej 30% w porównaniu z podobnym produktem. W 78 spośród 150 badanych produktów obniżono zawartość tłuszczu i/lub zastosowano jego zamienniki, natomiast w 54 produktach obniżono zawartość cukru i/lub zastosowano środki słodzące. Spośród wszystkich produktów 1/4 posiadała na opakowaniu oświadczenie o obniżonej kaloryczności i faktycznie spełniała jego warunki.Introduction: In accordance with Regulation EC No 1924/2006 the term „low in” including calories, it can be applied to products in which the decrease of the component was at least 30% compared to a similar product. In 78 products fat content was reduced and/or it replacement have been used, while in the 54 products, the energy-reduction was achieved by reducing sugar content and/or sweeteners were used. ¼ of all the products held on the package declaration about energy-reduction and actually meet the declaration conditions

Aktywność biologiczna bursztynu bałtyckiego – żywicy kopalnej

This paper constitutes a review concerning studies on bioactivity of Baltic amber. Baltic amber (succinite) – Eocene fossil resin – is a very complicated mixture consisting of polymeric and low molecular mass components. In folk medicine, succinite is thought to be a remedy for all ailments or diseases. However, there are no scientific results confirming the assumption that succinite can positively influence human body. To confirm it indirectly, many articles examining succinite components were analyzed. Basing on them, we realized that many properties attributed to Baltic amber may originate from its components also released from its polymeric structure. Observed properties are: antioxidative activity, antimicrobial activity, antiphlogistic activity, repellent and insecticidal activity – they coincide with folk medicine applications of succinite.Artykuł stanowi przegląd literatury dotyczącej badań bioaktywności bursztynu bałtyckiego. Bursztyn bałtycki (sukcynit) – eoceńska żywica kopalna – jest skomplikowaną mieszaniną polimerów i składników małocząsteczkowych. W medycynie ludowej sukcynit używany jest jako cudowny lek na wiele dolegliwości i chorób. Brak jednak naukowych dowodów potwierdzających pozytywny wpływ sukcynitu na organizm człowieka, które uzasadniałyby stosowanie preparatów z bursztynu np. w kosmetykach. Aby pośrednio sprawdzić zasadność doniesień ludowych, przeanalizowano artykuły dotyczące aktywności biologicznej składników bursztynu bałtyckiego zakładając, że właściwości bursztynu muszą wynikać zarówno z właściwości jego składników, jak również z ich umieszczenia w polimerycznej strukturze sukcynitu. Stwierdzono, że właściwości biologiczne składników bursztynu – antyoksydacyjne, przeciwdrobnoustrojowe, przeciwzapalne, repelencyjne oraz insektobójcze mogą być uzasadnieniem doniesień medycyny ludowej o stosowaniu bursztynu

The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding

The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli.

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop's region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections

Temperature-induced conformational changes within the regulatory loops L1-L2-LA of the HtrA heat-shock protease from Escherichia coli.

The present investigation was undertaken to characterize mechanism of thermal activation of serine protease HtrA (DegP) from Escherichia coli. We monitored the temperature-induced structural changes within the regulatory loops L1, L2 and LA using a set of single-Trp HtrA mutants. The accessibility of each Trp residue to aqueous medium at temperature range 25-45 degrees C was assessed by steady-state fluorescence quenching using acrylamide and these results in combination with mean fluorescence lifetimes (tau) and wavelength emission maxima (lambda(em)max) were correlated with the induction of the HtrA proteolytic activity. Generally the temperature shift caused better exposure of Trps to the quencher; although, each of the loops was affected differently. The LA loop seemed to be the most prone to temperature-induced conformational changes and a significant opening of its structure was observed even at the lowest temperatures tested (25-30 degrees C). To the contrary, the L1 loop, containing the active site serine, remained relatively unchanged up to 40 degrees C. The L2 loop was the most exposed element and showed the most pronounced changes at temperatures exceeding 35 degrees C. Summing up, the HtrA structure appears to open gradually, parallel to the gradual increase of its proteolytic activity