Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

l-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.List of Symbols HPLC High Performance Liquid Chromatography - HPTLC High Performance Thin Layer Chromatography - Trp tryptophan - Ser Serine - A amount of sorbent(g) - c equilibrium solute concentration in the aqueous phase (g/dm³) - c _i initial (before adding the sorbent) liquid phase concentration (g/dm³) - C _T tryptophan concentration in the inlet solution (g/dm³) - C _To tryptophan concentration in the outlet solution (g/dm³) - E _z axial dispersion coefficient (m²/s) - k experimental constant (Eq. 1, 2 and 3) - K ₁ rate constant of adsorption (min^–1) - L column length(m) - n experimental constant (eq. 1, 2 and 3) - q equilibrium solid phase concentration (g solute/g sorbent) - q _max maximum capacity of sorbent (g solute/g sorbent) - t time(s) - v liquid velocity (m/s) - V volume of liquid phase(dm³) - V _e eluted volume(dm³) - V _r volume needed to saturate the column (dm³)     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

Susceptibility of heterochromatin to aphidicolin-induced chromosomal breakage

The distribution of aphidicolin-induced chromosomal lesions was analyzed to determine the relative breakage susceptibility of euchromatin and heterochromatin in the cactus mouse, Peromyscus eremicus. The observed breakage was tested against expected distributions corresponding to the karyotypic proportions of autosomal euchromatin, autosomal heterochromatin, X-chromosomal euchromatin, and X-chromosomal heterochromatin. The distribution of induced breakage was independent of sex but dependent on the individual. In all individuals, there was a highly significant (P0.0001) deficiency in the number of breaks observed as compared to expected in autosomal heterochromatin. Sparse observations in the X chromosome and the absence of breaks in the Y chromosome precluded valid statistical tests of the sex-chromosomal distribution of induced breakage. These data indicate that the autosomal heterochromatin of Peromyscus is resistant to aphidicolin-induced chromosomal breakage and argue against a simple relationship between late replication and a general mechanism for chromosomal fragility.     

The expression of the salt-responsive gene salT from rice is regulated by hormonal and developmental cues

The expression pattern of the salT gene was analyzed in different cell types and organs of rice (Oryza sativa L.) in response to saline and hormonal treatments to obtain detailed information on the physiological cues controlling gene expression. Gel blot analysis of RNA and in-situ hybridization performed on seedlings grown for 10 ds in the presence of 1% NaCl revealed that salT was expressed mainly in the younger tissues of the plant. In contrast, 6-week-old plants exhibited maximal salT mRNA accumulation in sheaths of older leaves. In addition, salT was normally expressed in rapidly dividing suspension-cultured cells, but not in quiescent ones. Altogether, these results may indicate that salT expression in each region of the plant is dependent on the metabolic activity of the cells as well as on whether or not they are stressed. The effects of two growth regulators, abscisic acid (ABA) and gibberellic acid, were investigated in combination with the effects of NaCl. Gibberellic acid had a synergistic effect on the induction of the salT gene when combined with 0.5% NaCl, but did not induce salT on its own. At 10 μM, ABA induced salT both in the absence of NaCl and in its presence. Whereas 1 μM ABA acted additively with NaCl to induce gene expression, 5 μM ABA with NaCl was only as effective as NaCl alone. This may indicate that the two stimuli act independently and possibly through antagonistic signal transduction pathways. Received: 26 March 1998 / Accepted: 11 July 1998     

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.     

Genotoxic evaluation of the organophosphorous pesticide temephos

The chemical compound temephos (0,0,0',0'-tetrametyl-0,0'-thiodi-p-phenylene phosphorothioate) is an organophosphorous pesticide that has been used in Brazil since 1967 in control campaigns against the mosquito Aedes aegypti, the vector of dengue and yellow fever. We used single cell gel electrophoresis (SCGE), SOS/umu and Ames/Salmonella assays to test the toxicity and mutagenicity of temephos. Temephos was genotoxic in the SCGE assay, inducing severe DNA lesions (type IV lesions) at doses above 1.34 micro M. It was mutagenic, but not toxic, in the SOS/umu assay to Escherichia coli strain PQ37, but not to PQ35, at concentrations above 1.33 micro M, particularly when the S9 mixture was not used in the assay. Temephos was not mutagenic in the Ames assay with S. typhimurium strains TA97, TA98, TA100 and TA102, both with and without metabolic activation. However, temephos at concentrations above 3.33 micro M was mutagenic to TA98NR, YG7104 and YG7108, both with and without metabolic activation. In conclusion, temephos was genotoxic and mutagenic in all the three tests used, and in two of them at concentrations similar to those routinely used to combat Aedes aegypti.     

Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors

The involvement of nitric oxide (NO), prostaglandins, and calcium-dependent potassium channel (K(Ca)) activators on the negative modulation of phenylephrine-induced contractions was evaluated on the isolated aorta and caudal (CAU) artery obtained from rats treated with ouabain for 5 wk to induce hypertension. In ouabain-treated rats, the reactivity to phenylephrine was reduced in the endothelium-intact aorta but not the CAU segments. Endothelial modulation of phenylephrine contraction, as demonstrated by endothelium removal, NO synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester and aminoguanidine, as well as K(Ca) inhibition with tetraethylammonium, was more pronounced in segments from ouabain-treated animals, and here greater effects were seen in the aorta than in CAU. An increased expression of endothelial NOS and neuronal NOS was seen in the aorta after ouabain treatment. In CAU, only endothelial NOS was detected and ouabain treatment did not alter its expression. These results suggest that ouabain-induced hypertension is accompanied by increased NO release derived from endothelial NOS and neuronal NOS and increased release of an endothelial hyperpolarizing factor that presumably opens K(Ca), all of which contribute to the increased negative modulation of the phenylephrine contraction.     

Molecular evolution of key genes for type II secretion in Legionella pneumophila

Given the role of type II protein secretion system (T2S) in the ecology and pathogenesis of Legionella pneumophila, it is possible that this system is a target for adaptive evolution. The population genetic structure of L.pneumophila was inferred from the partial sequences of rpoB and from the complete sequence of three T2S structural components (lspD, lspE and pilD) and from two T2S effectors critical for intracellular infection of protozoa (proA and srnA) of 37 strains isolated from natural and man-made environments and disease-related from worldwide sources. A phylogenetic analysis was obtained for the concatenated alignment and for each individual locus. Seven main groups were identified containing the same L.pneumophila strains, suggesting an ancient divergence for each cluster and indicating that the operating selective pressures have equally affected the evolution of the five genes. Although linkage disequilibrium analysis indicate a clonal nature for population structure in this sample, our results indicate that recombination is a common phenomenon among T2S-related genes on this species, as 24 of the 37 L.pneumophila isolates contained at least one locus in which recombination was identified. Furthermore, neutral selection acting on the analysed T2S-related genes emerged as a clear result, namely on T2S effectors, ProA and SrnA, indicating that they are probably implicated in conserved virulence mechanisms through legionellae hosts.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.     

Relationships between IgE/IgG4 Epitopes,Structure and Function in Anisakis simplex Ani s 5, a Member of the SXP/RAL-2 Protein Family

Background

Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies.The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown.The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG₄ binding regions.

Methodology/Principal Findings

The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg²⁺, but not Ca²⁺, binding was determined by band shift using SDS-PAGE. IgE and IgG₄ epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients.The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG₄. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera.

Conclusions/Significance

This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG₄ binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.