Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum

<p>Abstract</p> <p>Background</p> <p>Laccases are multi-copper oxidases that catalyze the one electron oxidation of a broad range of compounds. Laccase substrates include substituted phenols, arylamines and aromatic thiols. Such compounds are activated by the enzyme to the corresponding radicals. Owing to their broad substrate range laccases are considered to be versatile biocatalysts which are capable of oxidizing natural and non-natural industrial compounds, with water as sole by-product.</p> <p>Results</p> <p>A novel CotA-type laccase from <it>Bacillus pumilus </it>was cloned, expressed and purified and its biochemical characteristics are presented here. The molecular weight of the purified laccase was estimated to be 58 kDa and the enzyme was found to be associated with four copper atoms. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine (SGZ) was investigated. The kinetic parameters <it>K</it>_Mand <it>k</it>_catfor ABTS were 80 ± 4 μM and 291 ± 2.7 s^-1, for 2,6-DMP 680 ± 27 μM and 11 ± 0.1 s^-1and for SGZ only <it>k</it>_catcould be estimated to be 66 ± 1.5 s^-1. The pH optimum for ABTS was 4, for 2,6-DMP 7 and for SGZ 6.5 and temperature optima for ABTS and 2,6-DMP were found to be around 70°C. The screening of 37 natural and non-natural compounds as substrates for <it>B. pumilus </it>laccase revealed 18 suitable compounds. Three of them served as redox mediators in the laccase-catalyzed decolorization of the dye indigocarmine (IC), thus assessing the new enzyme's biotechnological potential.</p> <p>Conclusions</p> <p>The fully copper loaded, thermostable CotA laccase from <it>Bacillus pumilus </it>is a versatile laccase with potential applications as an industrial biocatalyst.</p

Investigating the role of caspase cleavage of ROCK1 in tissue homeostasis and tumour development

During apoptosis, caspase cleavage of ROCK1 removes an auto-inhibitory region yielding a constitutively active kinase fragment. This results in phosphorylation of downstream targets that promote contractile force generation leading to cell shrinkage, membrane blebbing and nuclear disintegration. To address fundamental questions regarding the purpose of ROCK1 cleavage and consequent apoptotic morphological features, a novel mouse model was generated that carries a single amino acid substitution in the caspase cleavage site (D1113A) that converts ROCK1 to a caspase-resistant non-cleavable (ROCK1nc) form. When apoptosis was induced in ROCK1nc cells, morphological features were significantly impaired, although the biochemical apoptotic program itself was unaffected. To understand the biological role of apoptotic morphological features in the maintenance of tissue homeostasis, acute liver damage was induced in mice with the liver-selective genotoxic compound diethylnitrosamine (DEN). Following DEN treatment, there were increased TUNEL-positive apoptotic cells and increased neutrophil infiltration in ROCK1nc mice. Histologically, ROCK1nc livers were more damaged, paralleled by higher serum alanine transaminase levels. We hypothesized that uncleared apoptotic debris undergoing secondary necrosis may release damage associated molecular patterns (DAMPs) that may aggravate liver damage by recruiting neutrophils to the liver. Indeed, inhibiting the cytokine activities of HMGB1 reduced neutrophil infiltration as well as liver damage in ROCK1nc mice. Furthermore, to determine whether defects in tissue damage responses in ROCK1nc mice would affect tumour development, the ROCK1nc mutation was introduced into two different cancer models, specifically the DEN-induced hepatocellular carcinoma and Eµ-myc lymphoma mouse models. Defective caspase cleavage of ROCK1 promoted increased infiltration of CD8+ T-cells in ROCK1nc liver tumours and had a protective effect against tumour development in both tumour models. Taken together, our results indicate that apoptotic morphological features suppress inflammation which helps to maintain tissue homeostasis but enables tumourigenesis

Pengembangan Video Pembelajaran Berbasis Lingkungan untuk Meningkatkan Literasi Siswa SMP di Kelas VIII

This research was conducted to create an environment-based learning video that would help junior high school students in their literacy skills. In order to increase the literacy of junior high school students, this research aims to make environmental education films as cutting-edge media. Research and development techniques used in this study (Research and Development). To collect data from different people or groups, this study uses a questionnaire instrument consisting of a number of written questions. The results showed that when measured using environmental literacy indicators, students' environmental literacy levels had different average values, with cognitive skills of 80% (good criteria), environmental awareness of 70.67% (good criteria), and responsible behavior. environmental responsibility. of 89.22% (very good criteria)

A Cross-Cultural Comparison of Deradicalisation: Results from Germany and Pakistan

Deradicalisation refers to the process of distancing oneself from extremist ideologies. As a social challenge, it is usually addressed by specially qualified professionals. In this paper, based on 16 interviews with deradicalisation professionals, we comparatively examine deradicalisation practices in coercive environments in Germany and Pakistan. This cross-cultural comparison using “most dissimilar” cases allows us to distinguish between general and culture-specific approaches, while also allowing the strengths and weaknesses of the different approaches to emerge; this can be used to further develop deradicalisation efforts. Based on our evaluation of target groups, goals, professional understandings of radicalisation and its methods, this text elaborates the differences and similarities of deradicalisation practices; we also formulate consequences for deradicalisation practice and outline the need for further research

Impact of early environment on children's mental health : lessons from DNA methylation studies with monozygotic twins

Over the past decade, epigenetic analyses have made important contributions to our understanding of healthy development and a wide variety of adverse conditions such as cancer and psychopathology. There is increasing evidence that DNA methylation is a mechanism by which environmental factors influence gene transcription and, ultimately, phenotype. However, differentiating the effects of the environment from those of genetics on DNA methylation profiles remains a significant challenge. Monozygotic (MZ) twin study designs are unique in their ability to control for genetic differences because each pair of MZ twins shares essentially the same genetic sequence with the exception of a small number of de novo mutations and copy number variations. Thus, differences within twin pairs in gene expression and phenotype, including behavior, can be attributed in the majority of cases to environmental effects rather than genetic influence. In this article, we review the literature showing how MZ twin designs can be used to study basic epigenetic principles, contributing to understanding the role of early in utero and postnatal environmental factors on the development of psychopathology. We also highlight the importance of initiating longitudinal and experimental studies with MZ twins during pregnancy. This approach is especially important to identify: (1) critical time periods during which the early environment can impact brain and mental health development, and (2) the specific mechanisms through which early environmental effects may be mediated. These studies may inform the optimum timing and design for early preventive interventions aimed at reducing risk for psychopathology

Expression of a pathogenic mutation of SOD1 sensitizes aprataxin-deficient cells and mice to oxidative stress and triggers hallmarks of premature ageing

Aprataxin (APTX) deficiency causes progressive cerebellar degeneration, ataxia and oculomotor apraxia in man. Cell free assays and crystal structure studies demonstrate a role for APTX in resolving 5'-adenylated nucleic acid breaks, however, APTX function in vertebrates remains unclear due to the lack of an appropriate model system. Here, we generated a murine model in which a pathogenic mutant of superoxide dismutase 1 (SOD1(G93A)) is expressed in an Aptx-/- mouse strain. We report a delayed population doubling and accelerated senescence in Aptx-/- primary mouse fibroblasts, which is not due to detectable telomere instability or cell cycle deregulation but is associated with a reduction in transcription recovery following oxidative stress. Expression of SOD1(G93A) uncovers a survival defect ex vivo in cultured cells and in vivo in tissues lacking Aptx. The surviving neurons feature numerous and deep nuclear envelope invaginations, a hallmark of cellular stress. Furthermore, they possess an elevated number of high-density nuclear regions and a concomitant increase in histone H3 K9 trimethylation, hallmarks of silenced chromatin. Finally, the accelerated cellular senescence was also observed at the organismal level as shown by down-regulation of insulin-like growth factor 1 (IGF-1), a hallmark of premature ageing. Together, this study demonstrates a protective role of Aptx in vivo and suggests that its loss results in progressive accumulation of DNA breaks in the nervous system, triggering hallmarks of premature ageing, systemically

Production of glycoprotein vaccines in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. <it>In vivo </it>enzymatic coupling using the general glycosylation pathway of <it>Campylobacter jejuni </it>in recombinant <it>Escherichia coli </it>has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the <it>in vivo </it>biosynthesis of two novel conjugate vaccine candidates against <it>Shigella dysenteriae </it>type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.</p> <p>Results</p> <p>Two different periplasmic carrier proteins, AcrA from <it>C. jejuni </it>and a toxoid form of <it>Pseudomonas aeruginosa </it>exotoxin were glycosylated with <it>Shigella </it>O antigens in <it>E. coli</it>. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in <it>E. coli</it>. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of <it>E. coli </it>cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.</p> <p>Conclusions</p> <p>The presented data demonstrate that glycosylated proteins can be produced in recombinant <it>E. coli </it>at a larger scale. The described methodologies constitute an important step towards cost-effective <it>in vivo </it>production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.</p