Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.     

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.     

Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of β(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.     

Conversion of post consumer polyethylene to the biodegradable polymer polyhydroxyalkanoate

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

The role of 26S proteasome-dependent proteolysis in the formation and restructuring of microtubule networks

In this review, we summarize the evidence pointing at the important role of 26S proteasome-dependent proteolysis in the regulation of microtubule synthesis and microtubule dynamics. Because most of the advances in this relatively unexplored research field originate from yeast and animal studies, we have considered those studies that describe the role of proteolysis in processes that are evolutionarily conserved and known to exist in plants. In addition, we place particular emphasis on the proteasome-dependent degradation of plant-specific microtubule-associated protein SPIRAL1 and its function in MT rearrangements associated with salt stress.     

Neuroprotective Potential and Chemical Profile of Alternatively Cultivated Ganoderma lucidum Basidiocarps

Various neurodegenerative diseases are the main challenges to the modern medicine and there is a great need for novel, natural, neuroprotective agents. Ganoderma lucidum is a well‐known medicinal mushroom, which health benefits have been confirmed by numerous studies. As demand for its basidiocarps is increased and traditional cultivation on hardwoods is not environmentally friendly and economically justified, finding of alternative substrates is necessary. The aim of the study was to assess the effect of alternative cultivation substrates on the chemical profile of G. lucidum basidiocarps and their capacity to inhibit acetylcholinesterase and tyrosinase, which higher activity is directly associated with neurodegenerative processes. Extracts of basidiocarps cultivated on alternative substrates, especially on clear wheat straw, showed significantly higher inhibition capacities than extracts of commercially‐grown ones. These extracts were considerably different chemically from commercial basidiocarps extracts and even nine new compounds were isolated from them. Our results suggest that cultivation substrate greatly affect the chemical profile and neuroprotective capacity of obtained basidiocarps and wheat straw is a promising cultivation substrate.     

Exclusive cytosolic localization and broad tRNA<Superscript>Ser</Superscript> specificity of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seryl-tRNA synthetase

Aminoacyl-tRNA synthetases (aaRSs) decipher the genetic code, covalently linking amino acids to cognate tRNAs, thus preparing substrates for the process of translation. Although aaRSs funtion primarily in translation and are localized in cytosol, mitochondria and chloroplasts there are many reports on their additional functions and subcellular destinations beyond translation. However, data on plant aaRSs are scarce. Initial analysis of amino acid sequence of Arabidopsis thaliana seryl-tRNA synthetase (SerRS) suggested that protein contains putative nuclear localization signals. GFP-localization experiments in transiently transformed epidermal onion cells and Arabidopsis protoplasts gave ambiguous results because in some cells SerRS appeared to be dually localized to both cytosol and nucleus. However, data obtained on transgenic lines expressing SerRS-TAP and GFP-SerRS revealed exclusive cytosolic location of SerRS. Subcellular distribution of SerRS did not change during stress. Cytosolic Arabidopsis SerRS was expressed and purified. The enzyme efficiently aminoacylated eukaryotic and bacterial tRNAs^Ser, that are structurally very different. Given the fact that the same behavior was previously shown for monocot maize SerRS, it seems that plant SerRSs exhibit unusually broad tRNA^Ser specificity, unlike SerRSs from other organisms. Possible functional implications of this unique characteristic of plant SerRSs are discussed.     

Kinetic Analysis of Leucine-Enkephalin Cellular Uptake at the Luminal Side of the Blood-Brain Barrier of an In Situ Perfused Guinea-Pig Brain

Abstract: The uptake of enkephalin-(5-L-leucine) (Leu-en-kephalin) at the luminal side of the blood-brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, K_in, for [tyrosyl-3,5-³H]Leu-enkephalin was estimated graphically from the multiple-time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose-dependent self-inhibition was demonstrated. Analysis of unidirectional influx of blood-borne Leu-en kephalin into the brain revealed Michaelis-Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with V_max between 0.14 and 0.16 nmol min^?1 g^?1 and K_m ranging from 34 to 41 μM, for the saturable component, whereas the estimated diffusion constant, K_d, was not significantly different from zero. Entry of [³H]Leu-enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L-tyrosine, tyro-sylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood-brain barrier does not involve uptake of the peptide's N-terminal amino acid and/or its tyrosine-containing fragments. The specific δ-opioid antagonist, allyl²-Tyr-AIB-Phe-OH, and μ-opioid receptor agonist, Tyr-D-Ala-Gly-Me-Phe-NH(CH₂)20H, at concentrations in the perfusate above the K_m value for the saturable transport of Leu-enkephalin, did not affect significantly uptake of [³H]Leu-enkephalin. The present study provides, for the first time, a characterization of the kinetic parameters of the unidirectional uptake of a peptide from the luminal side of the blood-brain barrier