Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens

Adventitious shoots formed on the proximal cut edges of different cotyledonary explants of watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai; cvs. Sweet Gem and Gold Medal] cultured on Murashige and Skoog's (MS) medium with 1 mgl^-1 6-benzyladenine (BA). Light (16-h photoperiod, about 7 Wm^-2 cool-white fluorescent lamps) was essential for shoot formation. To obtain transformed plants, cotyledonary explants of Sweet Gem were cocultured with Agrobacterium tumefaciens LBA4404, a disarmed strain harboring a binary vector pBI121 carrying the CaMV 35S promoter--glucuronidase (GUS) gene fusion used as a reporter gene and NOS promoter-neomycin phosphotransferase gene as a positive selection marker, for 48 h on MS medium with 1 mgl^-1 BA and 200 M -hydroxyacetosyringone. After 48 h of culture, explants were transferred to medium with 1 mgl^-1 BA 250 mgl^-1 carbenicillin, and 100 mgl^-1 kanamycin and cultured in the light. Adventitious shoots formed on the explants after 4 weeks of culture. When subjected to GUS histochemical assay, young leaves obtained from the shoots showed a positive response at a frequency of up to 16%. Preculturing cotyledonary explants on MS medium with 1 mgl^-1 BA for 5 d enhanced the competence of the cells to be transformed by Agrobacterium. Southern blot analysis confirmed that the GUS gene was incorporated into the genomic DNA of the GUS-positive regenerants. The transformed plants were grown to maturity.     

Metabolic engineering of Mannheimia succiniciproducens for malic acid production using dimethylsulfoxide as an electron acceptor

Microbial production of various TCA intermediates and related chemicals through the reductive TCA cycle has been of great interest. However, rumen bacteria that naturally possess strong reductive TCA cycle have been rarely studied to produce these chemicals, except for succinic acid, due to their dependence on fumarate reduction to transport electrons for ATP synthesis. In this study, malic acid (MA), a dicarboxylic acid of industrial importance, was selected as a target chemical for mass production using Mannheimia succiniciproducens, a rumen bacterium possessing a strong reductive branch of the TCA cycle. The metabolic pathway was reconstructed by eliminating fumarase to prevent MA conversion to fumarate. The respiration system of M. succiniciproducens was reconstructed by introducing the Actinobacillus succinogenes dimethylsulfoxide (DMSO) reductase to improve cell growth using DMSO as an electron acceptor. Also, the cell membrane was engineered by employing Pseudomonas aeruginosa cis-trans isomerase to enhance MA tolerance. High inoculum fed-batch fermentation of the final engineered strain produced 61 g/L of MA with an overall productivity of 2.27 g/L/h, which is the highest MA productivity reported to date. The systems metabolic engineering strategies reported in this study will be useful for developing anaerobic bioprocesses for the production of various industrially important chemicals.     

iBench: A ground truth approach for advanced validation of mass spectrometry identification method

The discovery of many noncanonical peptides detectable with sensitive mass spectrometry inside, outside, and on cells shepherded the development of novel methods for their identification, often not supported by a systematic benchmarking with other methods. We here propose iBench, a bioinformatic tool that can construct ground truth proteomics datasets and cognate databases, thereby generating a training court wherein methods, search engines, and proteomics strategies can be tested, and their performances estimated by the same tool. iBench can be coupled to the main database search engines, allows the selection of customized features of mass spectrometry spectra and peptides, provides standard benchmarking outputs, and is open source. The proof-of-concept application to tryptic proteome digestions, immunopeptidomes, and synthetic peptide libraries dissected the impact that noncanonical peptides could have on the identification of canonical peptides by Mascot search with rescoring via Percolator (Mascot+Percolator).     

ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

In N-limited continuous chemostat cultures of the green alga Haematococcus lacustris (Gir.) Rostaf. (UTEX 16), the steady-state astaxanthin content of the cells was determined by the specific growth rate of the cultures. The highest, pigment content was obtained at the lowest dilution rate. The specific rate of astaxanthin accumulation was, however, a function of the photon flux density measured at the illuminated culture surface. In nongrowing Haematococcus cultures, the specific rate of astaxanthin accumulation was determined by the growth rate of the culture during growth phase. The highest possible cellular astaxanthin content of all cultures was comparable and independent of the culture parameters.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Modulation of cortisol receptors in embryonic retina cells by changes in cell-cell contacts: Correlations with induction of glutamine synthetase

Cortisol induces glutamine synthetase (GS) in neural retina tissue of chick embryos. GS induction represents a characteristic feature of embryonic retina differentiation. However, if the tissue is dissociated into single cells, the dispersed cells are not inducible for GS. We report that cell dispersion results in a rapid and marked reduction in the level of cortisol-binding cytoplasmic receptors. This reduction persists if the cells are maintained in a dispersed state. However, if the cells are reaggregated and they reconstruct tissue-like contacts and architecture, the level of cortisol receptors increases, and so does inducibility for GS. The results indicate that, in the embryonic neural retina histotypic cell contacts and interactions are involved in regulating the level of cortisol receptors. We propose that cell contact-dependent signals from the cell surface may modulate levels of cytoplasmic cortisol receptors necessary for GS induction.     

Synthesis and some reactions of 3,5-di-O-methyl-d-glucose and -fructose

Hydrolysis of 1,2-O-isopropylidene-3,5-di-O-methyl-α-d-glucofuranose by strong acid yielded 3,5-di-O-methyl-d-glucofuranose (6) and its 1,6-anhydride (10). The mechanism of the reaction giving 10 is discussed. On treatment with a catalytic amount of sodium methoxide, 1,2,6-tri-O-acetyl-3,5-di-O-methyl-d-glucofuranose (8) gives the 6-O-acetyl derivative, whereas complete deacetylation, and subsequent isomerization to the d-fructose derivative 16, takes place in the presence of 0.1m sodium methoxide. The structure of 16 was proved both chemically and spectroscopically. Reduction of 6 or 8 with a borohydride afforded 3,5-di-O-methyl-d-glucitol.²     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.