Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica

The production of benzophenanthridine alkaloids (sanguinarine, chelerythrine and macarpine) in cells of Eschscholtzia californica is enhanced by sodium alginate and by entrapment in Ca2+-alginate. Tyrosine decarboxylase, a key enzyme of alkaloid biosynthesis, is induced by the treatments. Alginate- entrapped cells are elicited over an extended period of time which leads to increased alkaloid biosynthesis (up to 800-fold enhancement). A major portion of alkaloids produced are released into the growth medium.     

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.     

Efficient access to all four stereoisomers of phenylalanine cyclopropane analogues by chiral HPLC

Bonded polysaccharide‐derived chiral stationary phases were found to be useful for the preparation of the four stereoisomers of the cyclopropane analogue of phenylalanine (c₃Phe) as well as for the direct determination of the enantiomeric purity of c₃Phe derivatives by HPLC. Three chiral stationary phases, consisting of cellulose and amylose derivatives chemically bonded on allylsilica gel, were tested. The mixed 10‐undecenoate/3,5‐dimethylphenylcarbamate of cellulose, 10‐undecenoate/3,5‐dimethylphenylcarbamate of amylose and 10‐undecenoate/p‐methylbenzoate of cellulose were the starting polysaccharide derivatives for CSP‐1, CSP‐2, and CSP‐3, respectively. Using mixtures of n‐hexane/chloroform/2‐propanol as mobile phase on a semi‐preparative column (150 mm × 20 mm ID) containing CSP‐2, we separated about 1.7 g of racemic cis‐methyl 1‐tert‐butoxycarbonylamino‐2‐phenylcyclopropanecarboxylate (cis‐ 6 ) and 1.2 g of racemic trans‐methyl‐1‐tert‐butoxycarbonylamino‐2‐phenylcycloprop‐anecarboxylate (trans‐ 6 ) by successive injections. Chirality 11:583–590, 1999. © 1999 Wiley‐Liss, Inc.     

Expression profiling identifies novel Hh/Gli-regulated genes in developing zebrafish embryos

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Misregulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants that affect different components of the Hh/Gli signaling system have been identified. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified known (e.g., ptc1 and nkx2.2a) and novel Hh-regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue-specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli-regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of misregulation during tumorigenesis.     

Synthesis, Solution Structure, and Phylum Selectivity of a Spider ��-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na_V) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na_V channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na_V channel subtypes showing that δ-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na_V1.1, Na_V1.3, and Na_V1.6 but more importantly shows higher affinity for insect Na_V1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect Na_V channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.Voltage-gated sodium (Na_V)⁴ channels are responsible for the generation and propagation of electrical signals in excitable cells. At least nine different genes encoding distinct Na_V channels isoforms have been identified, and functionally expressed, in mammals (1). They are characterized by their sensitivity to TTX, with Na_V1.5, Na_V1.8, and Na_V1.9 being TTX-insensitive or TTX-resistant, and the remaining subtypes being sensitive to nanomolar concentrations of TTX. In addition, localization of the subtypes also varies, with Na_V1.1–1.3 mostly distributed in the central nervous system, Na_V1.6–1.9 principally located in the peripheral nervous system, and Na_V1.4 and Na_V1.5 found in skeletal and cardiac muscle, respectively. The structural diversity of Na_V channels also coincides with variations in physiological and pharmacological properties (2). In contrast, insects express only one gene (para) that undergoes extensive alternative splicing and RNA editing (3). The para-encoded Na_V channel is exceptionally well conserved across diverse orders of insects, with the level of identity ranging from 87 to 98% (3). This is one reason why insecticides that target insect Na_V channels have broad activity across many insect orders. In contrast, para-type Na_V channels have significantly lower levels of identity with the various types of mammalian Na_V channels with the level of identity typically around 50–60% (3). This explains why a high degree of phylogenetic specificity can be achieved with both Na_V channel toxins and insecticides that target the Na_V channel.At least seven distinct toxin-binding sites have been identified by radioligand binding and electrophysiological studies on vertebrate and insect Na_V channels (4, 5). Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of Na_V channel topology, function, and pharmacology (6). In particular, a wide range of scorpion α-toxins, sea anemone toxins, and spider δ-hexatoxins (formerly δ-atracotoxins (7)) compete for binding to receptor site-3 on the extracellular surface of Na_V channels. These polypeptide toxins all inhibit the fast inactivation of Na_V channels to prolong Na⁺ currents (I_Na), despite huge diversity in primary and tertiary structures (8, 9). Nevertheless, receptor site-3 has not yet been fully characterized but is believed to involve domains DI/S5-S6, DIV/S5-S6, as well as DIV/S3-S4 (9). Most importantly, however, toxin characterization is often limited to studies using whole-cell I_Na or binding studies on neuronal membranes where there are mixed populations of Na_V channel subtypes. For all of these toxins, the precise pattern of Na_V channel subtype selectivity is either unknown or at best is incomplete.Recently, it was found that receptor site-3 was also recognized by a 43-residue spider toxin, originally named Magi 4, from the hexathelid spider Macrothele gigas (Iriomote, Japan). It binds with high affinity to insect Na_V channels but, similar to scorpion α-like toxins, does not compete for the related site-3 in rat brain synaptosomes, despite being lethal by intracranial injection (10). Magi 4 shares significant homology to four δ-hexatoxin (HXTX)-1 family peptides and δ-actinopoditoxin-Mb1a (formerly δ-missulenatoxin-Mb1a; Fig. 1) but no sequence homology to scorpion α-toxins. Neurochemical studies have shown that δ-HXTX-1 toxins compete at nanomolar concentrations with both anti-mammalian (e.g. Aah2 and Lqh2) and anti-insect (e.g. LqhαIT) scorpion toxins for site-3 (11–13). The three-dimensional structures of δ-HXTX-Ar1a and δ-HXTX-Hv1a peptides have been determined (14, 15) and possess core β regions stabilized by four disulfide bonds, placing them in the inhibitory cystine knot (ICK) structural family (16).Open in a separate windowFIGURE 1.Primary and secondary structure of δ-HXTX-Mg1a. A, comparison of the primary sequence of δ-HXTX-Mg1a and δ-HXTX-Mg1b (formerly Magi 14) with currently known members of the δ-HXTX-1 family and δ-AOTX-Mb1a (δ-actinopoditoxin-Mb1a, formerly δ-missulenatoxin-Mb1a). Homologies are shown relative to δ-HXTX-Mg1a; identities are boxed in gray, and conservative substitutions are in gray italic text. Gaps (dashes) have been inserted to maximize alignment. The disulfide bonding pattern for the strictly conserved cysteine residues determined for δ-HXTX-Mg1a (this study), δ-HXTX-Ar1a (55), and δ-HXTX-Hv1a (15) is indicated above the sequences; it is assumed that δ-AOTX-Mb1a (36), δ-HXTX-Hs20.1a (8), and δ-HXTX-Hv1b (56) have the same disulfide bonding pattern. The percentage identity and homology with δ-HXTX-Mg1a is shown to the right of the sequences. B, summary of δ-HXTX-Mg1a NMR data. Sequential NOEs, classified as very weak, weak, medium, and strong, are represented by the thickness of bars. Filled diamonds indicate backbone amide protons that form hydrogen bonds. ³J_NHCα coupling constants are indicated by ↑ (>8 Hz) and ↓ (<5.5 Hz). Secondary structure is shown at the bottom of the figure where rectangles represent β-turns (the type of turn is indicated in the rectangle) and arrows represent β-sheets.The aim of this study was to first determine the solution structure of Magi 4 and second to investigate the ability of Magi 4 to discriminate between different Na_V channels subtypes. Here we report the tertiary structure of Magi 4 by ¹H NMR and show its disulfide bonding pattern and three-dimensional structure are homologous to δ-HXTX-1 toxins. We highlight the key residues in Magi 4 that appear to be topologically similar to those residues known to be part of the pharmacophore for site-3 scorpion α-toxins, despite Magi 4 having a different overall structure to scorpion α-toxins (11). In addition, we provide a detailed characterization of the selectivity and mode of action of Magi 4 on nine cloned mammalian and insect Na_V channel subtypes, including a detailed characterization on insect neurotransmission. Given that the toxin potently slows the inactivation of Na_V channels, it should be renamed δ-hexatoxin-Mg1a (δ-HXTX-Mg1a) in accordance with the rational nomenclature recently proposed for naming spider peptide toxins (7) (see ArachnoServer spider toxin data base).     

Pax‐6 is expressed early in the differentiation of a corneal epithelial model system

Pax‐6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1‐(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax‐6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax‐6‐positive cells were also K3 keratin‐positive, suggesting that Pax‐6 is expressed in differentiating cells. Pax‐6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT‐qPCR. The raise in Pax‐6 expression preceded for 9 h the increase in LDH‐H and LDH‐M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full‐length mRNAs encoding for the two major Pax‐6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax‐6 mRNA was 2‐ to 2.5‐fold more abundant than Pax‐6(5a) mRNA. The ectopic expression of Pax‐6 or Pax‐6(5a) decreased proliferative ability leading to the formation of abortive, non‐proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax‐6. Collectively, results show that Pax‐6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax‐6 as the main driver of the differentiation of corneal epithelial cells. J. Cell. Physiol. 220: 348–356, 2009. © 2009 Wiley‐Liss, Inc.     

LuMPIS – A modified luminescence‐based mammalian interactome mapping pull‐down assay for the investigation of protein–protein interactions encoded by GC‐low ORFs

The GC content is highly variable among the genomes of different organisms. It has been shown that recombinant gene expression in mammalian cells is much more efficient when GC‐rich coding sequences of a certain protein are used. In order to study protein–protein interactions in Varicella zoster virus, a GC‐low herpesvirus, we have developed a novel luminescence‐based maltose‐binding protein pull‐down interaction screening system (LuMPIS) that is able to overcome the impaired protein expression levels of GC‐low ORFs in mammalian expression systems.     

Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

Background  

The pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq.     

Multi-Country Evaluation of the Sensitivity and Specificity of Two Commercially-Available NS1 ELISA Assays for Dengue Diagnosis

Background

Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis.

Methodology/Principal Findings

The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia™ Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity).

Conclusions/Significance

Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34–76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.