Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix

The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.     

Enhanced vincamine production in selected tryptophan-overproducing shoots of Vinca minor

Vinca minor is the sole source of vincamine, an alkaloid known to be used in a variety of cerebral disorders. Three stable variant shoot lines (V10, V20 and V30) with tolerance thresholds of 10, 20 and 30?mg/l 5-methyltryptophan (5-MT; analogue of tryptophan), respectively, were selected. These lines showed twofold to threefold increase in tryptophan content and 1.5- to 2-fold increment in the total alkaloids in comparison to the wild line shoots. A maximum of 16-fold enhancement in vincamine production was recorded in V30 line followed by eightfold in V20 line. Inter simple sequence repeat (ISSR)-PCR amplification of all the three lines showed total of 65 bands; out of which 60 were monomorphic (92.3?%) and 5 were polymorphic (7.7?%). Tryptophan being a limiting factor in the indole alkaloid pathway plays a crucial role in modulating the flux towards vincamine production and its over-production positively resulted into enhanced vincamine production.     

Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.     

New Frame Shift Mutation in Puroindoline B in Indian Wheat Cultivars Hyb65 and NI5439

Puroindoline genes pinA and pinB are the main components of the 15 kD friabilin protein reported to be associated with kernel softness. However, grain hardness of Hyb65 and NI5439, the two Indian wheat varieties, could not be explained based on the earlier identified alleles in puroindolines in wheat. Hyb65 and NI5439 are hard but based on the earlier identified allelic forms of puroindolines both the varieties could have been soft. In this investigation, puroindolines (a and b) from Hyb65 and NI5439 were characterised to understand their role in determining grain hardness. The sequence of puroindoline genes from both the varieties indicated that there was no mutation in pinA. However, there was frame shift mutation in pinB generated by insertion of a guanine residue 126 bp downstream from the start codon in both the varieties. This created new hardness allele of pinB designated as pinb-D1h. Frame shift also culminated into stop codon (TGA) 231 bp downstream from the start codon terminating protein synthesis at 77^th amino acid position. Five more stop codons (4TGA & 1TAG) were also created to the downstream positions of the first stop codon because of frame shift. There was additional point mutation in NI5439 (transition from A to G) resulting into change of amino acid residue from thymine to arginine at 205^th nucleotide position. Thus single nucleotide change in pinB resulted into truncated pin B and consequently the harder texture.     

Phospholipase-mediated preparation of 1-ricinoleoyl-2-acyl-sn- glycero-3-phosphocholine from soya and egg phosphatidylcholine

1-Ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine was prepared by incorporating ricinoleic acid completely in the sn-1 position of egg and soya phosphatidylcholine (PC) using immobilized phospholipase A₁ as the catalyst. The optimum reaction conditions for maximum incorporation of ricinoleic acid into PC through transesterification were 10% (w/w) immobilized enzyme (116 mg), a 1:5 mol ratio of PC (soya, 387 mg; egg, 384 mg) to methyl ricinoleate (780 mg) at 50 °C for 24 h in hexane.     

Silkworm nucleotide databases--current trends and future prospects

The domesticated silkworm, Bombyx mori serves as an ideal representative of lepidopteran species for a variety of scientific studies. As a result, databases have been created to organize information pertaining to the silkworm genome that is subject to constant updating. Of these, four main databases are important for store nucleotide information in the form of genomic data, ESTs and microsatelites. These databases also store data related to other lepidoptera and important insects, which help in insect biological research. Though a considerable amount of nucleotide data is currently available, there is a paucity of data related to silkworm and other lepidopteran proteins. Hence, the focus of this article is to present the current status of nucleotide databases of silkworm, avenues for improvement and possibilities for databases that could be created in the future.     

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.