Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: Release of the products as free fatty acids

In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg²⁺ to in vitro assays facilitates appearance of radiolabel from ¹⁴C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.     

First morphogenetic identification of <i>Fusarium solani</i> isolated from orange fruit in Egypt

Losses due to postharvest decay may occur at any time during postharvest handling, from harvest to consumption affecting the produce quality and quantity. Accurate identification of the pathogen causing postharvest disease is essential to the selection of an appropriate disease control approach. Nine isolates of Fusarium recovered from orange fruit were identified as Fusarium solani. The fungus is involved with fruit decay. The obtained cultures were purified and grown on potato-dextrose agar (PDA), malt yeast agar (MYA), and Czapek's nutrient media (CNM) under light for identification. A pathogenicity test was carried out to fulfil Koch's postulates. The pathogen could only enter ripe orange fruit through wounds and cracks causing the rot disease. The identification of the fungal isolates was confirmed to be F. solani by DNA sequencing, which was 99 to 100% homologous to those deposited in the Gen- Bank. The identity of nine fungal isolates was confirmed to be F. solani by DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank Accession Nos. DQ486874 to DQ486881 and KC758879). To our knowledge, this is the first morphogenetic identification of F. solani isolated from orange fruit in Egypt.     

Genetic differentiation,hybridization and adaptive divergence in two subspecies of the acorn barnacle Tetraclita japonica in the northwestern Pacific

Two acorn barnacles, Tetraclita japonica japonica and Tetraclita japonica formosana, have been recently reclassified as two subspecies, because they are morphologically similar and genetically indistinguishable in mitochondrial DNA sequences. The two barnacles are distinguishable by parietes colour and exhibit parapatric distributions, coexisting in Japan, where T. j. formosana is very low in abundance. Here we investigated the genetic differentiation between the subspecies using 209 polymorphic amplified fragment length polymorphism markers and 341 individuals from 12 locations. The subspecies are genetically highly differentiated (Φ_CT = 0.267). Bayesian analysis and principal component analysis indicate the presence of hybrids in T. j. formosana samples from Japan. Strong differentiation between the northern and southern populations of T. j. japonica was revealed, and a break between Taiwan and Okinawa was also found in T. j. formosana. The differentiation between the two taxa at individual loci does not deviate from neutral expectation, suggesting that the oceanographic pattern which restricts larval dispersal is a more important factor than divergent selection in maintaining genetic and phenotypic differentiation. The T. j. formosana in Japan are probably recent migrants from Okinawa, and their presence in Japan may represent a poleward range shift driven by global warming. This promotes hybridization and might lead to a breakdown of the boundary between the subspecies. However, both local adaptation and larval dispersal are crucial in determining the population structure within each subspecies. Our study provides new insights into the interplay of local adaptation and dispersal in determining the distribution and genetic structure of intertidal biota and the biogeography of the northwestern Pacific.     

Structure of the human Lanosterol Synthase gene and its analysis as a candidate for holoprosencephaly (HPE1)

Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.     

Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.     

A loss-of-function mutation in the CFC domain of TDGF1 is associated with human forebrain defects

TDGF1 (CRIPTO) is an EGF-CFC family member and an obligate co-receptor involved in NODAL signaling, a developmental program implicated in midline, forebrain, and left-right axis development in model organisms. Previous studies of CFC1 (CRYPTIC), another member of the EGF-CFC family, demonstrated that normal function of this protein is required for proper laterality development in humans. Here we identify a mutation in the conserved CFC domain of TDGF1 in a patient with midline anomalies of the forebrain. The mutant protein is inactive in a zebrafish rescue assay, indicating a role for TDGF1 in human midline and forebrain development.     

Mutations in PATCHED-1, the receptor for SONIC HEDGEHOG, are associated with holoprosencephaly

Holoprosencephaly (HPE) is the most commonly occurring congenital structural forebrain anomaly in humans. HPE is associated with mental retardation and craniofacial malformations. The genetic causes of HPE have recently begun to be identified, and we have previously shown that HPE can be caused by haploinsufficiency for SONIC HEDGEHOG ( SHH). We hypothesize that mutations in genes encoding other components of the SHH signaling pathway could also be associated with HPE. PATCHED-1 (PTCH), the receptor for SHH, normally acts to repress SHH signaling. This repression is relieved when SHH binds to PTCH. We analyzed PTCH as a candidate gene for HPE. Four different mutations in PTCHwere detected in five unrelated affected individuals. We predict that by enhancing the repressive activity of PTCH on the SHH pathway, these mutations cause decreased SHH signaling, and HPE results. The mutations could affect the ability of PTCH to bind SHH or perturb the intracellular interactions of PTCH with other proteins involved in SHH signaling. These findings further demonstrate the genetic heterogeneity associated with HPE, as well as showing that mutations in different components of a single signaling pathway can result in the same clinical condition.