MORPHOLOGY OF THE MARINE DIATOM NITZSCHIA NAVIS-VARINGICA, SP. NOV. (BACILLARIOPHYCEAE), ANOTHER PRODUCER OF THE NEUROTOXIN DOMOIC ACID

A new marine diatom, Nitzschia navis-varingica , sp. nov., isolated from Vietnamese waters, is described by light, transmission, and scanning electron microscopy, including thin sectioning. The new species has been found to produce the neurotoxin domoic acid (DA), better known from several species of Pseudo-nitzschia Peragallo and one species of Amphora Ehrenberg. Production of DA is therefore more widespread among diatoms than previously thought. Taxonomically, the genus Nitzschia Hassall is exceptionally difficult, with about 900 described taxa. Grunow (in Cleve and Grunow 1880 divided the genus into 24 sections, and this system is still used with modifications. Nitzschia navis-varingica , sp. nov. fits best into a group of sections that includes Dubiae, Bilobatae , most of the Lanceolatae , and Lineares , all sensu Grunow, as the cell is slightly indented in the middle in girdle view and has a moderately eccentric raphe and a weak longitudinal fold on the valve. Many species within these sections have features similar to N. navis-varingica , but no species seems to be identical. Because both Pseudo-nitzschia and Nitzschia belong to the family Bacillariaceae, it seems reasonable to look for further producers of DA in this family, including freshwater species, which mainly comprise species within the sections Dubiae, Bilobatae, Lanceolatae , and Lineares.     

PSEUDO-NITZSCHIA SPECIES (BACILLARIOPHYCEAE) IN LOUISIANA COASTAL WATERS: MOLECULAR PROBE FIELD TRIALS, GENETIC VARIABILITY, AND DOMOIC ACID ANALYSES

An 18-month field survey of the Pseudo-nitzschia population present in Louisiana coastal waters was conducted comparing species abundance estimates by novel fluorescent molecular probes (16S large subunit rDNA oligonucleotide sequences) with traditional electron and differential-interference light microscopy. While the probe and microscopic analyses agreed on the presence or absence of four common Pseudo-nitzschia species ( P. multiseries (Hasle) Hasle, P. pseudodelicatissima (Hasle) Hasle, P. delicatissima (P.T. Cleve) Heiden, and P. pungens (Grunow) Hasle in 66% of the samples analyzed, the probes gave conflicting results with the microscopic methods in the remaining 34% of the samples. The majority of the discrepancies appear to be because of genetic variation within the Pseudo-nitzschia population, especially in P. pseudodelicatissima, indicating that the Monterey Bay Pseudo-nitzschia spp. may not be appropriate reference strains for distinguishing Louisiana Pseudo-nitzschia spp. Additionally, P. pseudodelicatissima has been associated with domoic acid (DA) activity in three field samples, at levels up to 22 times higher than the highest value given inother published reports of DA production by this species. The contemporaneous existence of multiple strains of P. pseudodelicatissima (toxic and non-toxic) presents new challenges to the study of the ecophysiology and population dynamics of this bloom-forming species.     

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.     

Analysis and identification of essential genes in humans using topological properties and biological information

Genes that are indispensable for survival are termed essential genes. The analysis and identification of essential genes are very important for understanding the minimal requirements of cellular survival and for practical purposes. Proteins do not exert their function in isolation of one another but rather interact together in PPI networks. A global analysis of protein interaction networks provides an effective way to elucidate the relationships between proteins. With the recent large-scale identifications of essential genes and the production of large amounts of PPIs in humans, we are able to investigate the topological properties and biological properties of essential genes. However, until recently, no one has ever investigated human essential genes using topological and biological properties. In this study, for the first time, 28 topological properties and 22 biological properties were used to investigate the characteristics of essential and non-essential genes in humans. Most of the properties were statistically discriminative between essential and non-essential genes. The F-score was used to estimate the essentiality of each property. The GO-enrichment analysis was performed to investigate the functions of the essential and non-essential genes. Finally, based on the topological features and the biological characteristics, a machine-learning classifier was constructed to predict the essential genes. The results of the jackknife test and 10-fold cross validation test are encouraging, indicating that our classifier is an effective human essential gene discovery method.     

Construction and engineering of a thermostable self-sufficient cytochrome P450

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP⁺ reductase (FNR): H₂N-CYP175A1-Fdx-FNR-COOH (175FR) and H₂N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V_max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k_m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 °C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.     

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25 °C. The activity constants (k_cat) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25 °C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70 °C. The rate of catalysis at temperatures above ~ 70 °C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~ 5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.     

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.     

Delayed caspase-8 activation and enhanced integrin β1-activated FAK underpins anoikis in oesophageal carcinoma cells harbouring mt p53-R175H

FAK (focal adhesion kinase)-mediated signalling reportedly suppresses caspase-8 activation and, as a consequence, rescues epithelial cells from Fas-mediated anoikis. Critical was the use of a HOSCC (human oesophageal squamous carcinoma) cell line harbouring mt (mutant) p53-R175H and displaying resistance to detachment and Tyr397 dephosphorylation of FAK. Here we show, although caspase-8 activation is delayed in the mt p53-R175H cell line, comparable apoptotic events evidenced in the wt (wild type) p53 HOSCC cell lines could be induced in the mt p53-R175H cell line by strengthening the apoptotic stimulus. Significant to anoikis-related regulation, the delay in caspase-8 activation was accompanied by the maintenance of FAK Tyr397 phosphorylation, integrin β1-associated FAK and a FAK/caspase-8 complex. Thus, mt p53-R175H may desensitize tumours to Fas-mediated anchorage-independent death via a FAK-dependent mechanism.     

The hidden potential of lysosomal ion channels: A new era of oncogenes

Lysosomes serve as the control centre for cellular clearance. These membrane-bound organelles receive biomolecules destined for degradation from intracellular and extracellular pathways; thus, facilitating the production of energy and shaping the fate of the cell. At the base of their functionality are the lysosomal ion channels which mediate the function of the lysosome through the modulation of ion influx and efflux. Ion channels form pores in the membrane of lysosomes and allow the passage of ions, a seemingly simple task which harbours the potential of overthrowing the cell’s stability. Considered the master regulators of ion homeostasis, these integral membrane proteins enable the proper operation of the lysosome. Defects in the structure or function of these ion channels lead to the development of lysosomal storage diseases, neurodegenerative diseases and cancer. Although more than 50 years have passed since their discovery, lysosomes are not yet fully understood, with their ion channels being even less well characterized. However, significant improvements have been made in the development of drugs targeted against these ion channels as a means of combating diseases. In this review, we will examine how Ca²⁺, K⁺, Na⁺ and Cl⁻ ion channels affect the function of the lysosome, their involvement in hereditary and spontaneous diseases, and current ion channel-based therapies.     

Insights Into Pigmentary Phenomena Provided by Grafting and Chimera Formation in the Axolotl

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, and grafts of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.