A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Collagen XVI is expressed by human dermal fibroblasts and keratinocytes and is associated with the microfibrillar apparatus in the upper papillary dermis.

Indirect immunofluorescence staining of normal skin with affinity-purified antibodies revealed a conspicuous presence of collagen XVI at the dermo-epidermal interface where it occurs in close vicinity to collagen VII. In addition, the protein co-localizes with fibrillin 1 at the cutaneous basement membrane zone and the adjacent papillary dermis, but not in deeper layers of the dermis. Both fibronectin and collagen XVI are distributed throughout smooth muscles of hair follicles but do not co-localize. These data suggest, therefore, that collagen XVI contributes to the structural integrity of the dermo-epidermal junction zone by interacting with components of the anchoring complexes and the microfibrillar apparatus. A strong immunofluorescence signal associated with the extracellular matrix of individual cells was observed for keratinocytes or fibroblasts in monolayer cultures. Therefore, both cell types are likely sources of the protein also in situ. The rate of expression of collagen XVI mRNA in keratinocytes is about half of that in normal human skin fibroblasts. In both cell types, TGF-beta2 treatment results in an up-regulation of the collagen XVI-mRNA by approximately 50%. In keratinocytes, synthesis of collagen XVI protein and deposition to the cell layer and the extracellular matrix is stimulated fivefold and twofold, respectively. Since TGF-beta2 also upregulates the biosynthesis of other matrix macromolecules in the subepidermal zone the factor is likely to contribute to the stabilization of matrix zones near basement membranes in healing wounds.     

A novel regeneration system for a wild passion fruit species (<Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.     

Inhibition of advanced glycation end product formation on collagen by rutin and its metabolites

Several lines of evidence suggest that rutin, flavonoid in fruits and vegetables, or one of its metabolites may effectively modulate advanced glycation end product (AGE) formation. Following ingestion, rutin forms metabolites that include 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3,4-dihydroxytoluene (3,4-DHT), m-hydroxyphenylacetic acid (m-HPAA), 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid, HVA) and 3,5,7,3',5'-pentahydroxyflavonol (quercetin). We studied the effects of rutin and its metabolites on the formation of AGE biomarkers such as pentosidine, collagen-linked fluorescence, N(epsilon)-carboxymethyllysine (CML) adducts, glucose autoxidation and collagen glycation, using an in vitro model where collagen I was incubated with glucose. Rutin metabolites containing vicinyl dihydroxyl groups, i.e., 3,4-DHT, 3,4-DHPAA and quercetin, inhibited the formation of pentosidine and fluorescent adducts, glucose autoxidation and glycation of collagen I in a dose-dependent manner, whereas non-vicinyl dihydroxyl group-containing metabolites, i.e., HVA and m-HPAA, were much less effective. All five metabolites of rutin effectively inhibited CML formation. In contrast, during the initial stages of glycation and fluorescent AGE product accumulation, only vicinyl hydroxyl group-containing rutin metabolites were effective. These studies demonstrate that rutin and circulating metabolites of rutin can inhibit early glycation product formation, including both fluorescent and nonfluorescent AGEs induced by glucose glycation of collagen I in vitro. These effects likely contribute to the beneficial health effects associated with rutin consumption.     

Caribbean corals in crisis: record thermal stress, bleaching, and mortality in 2005

Background

The rising temperature of the world''s oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin.

Methodology/Principal Findings

Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers'' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles.

Conclusions/Significance

Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch''s Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.     

Translationally controlled tumour protein (TCTP) from tomato and Nicotiana benthamiana is necessary for successful infection by a potyvirus

Translationally controlled tumour protein (TCTP) is a ubiquitously distributed protein in eukaryotes, involved in the regulation of several processes, including cell cycle progression, cell growth, stress protection, apoptosis and maintenance of genomic integrity. Its expression is induced during the early stages of tomato (Solanum lycopersicum) infection by the potyvirus Pepper yellow mosaic virus (PepYMV, a close relative of Potato virus Y). Tomato TCTP is a protein of 168 amino acids, which contains all the conserved domains of the TCTP family. To study the effects of TCTP silencing in PepYMV infection, Nicotiana benthamiana plants were silenced by virus‐induced gene silencing (VIGS) and transgenic tomato plants silenced for TCTP were obtained. In the early stages of infection, both tomato and N. benthamiana silenced plants accumulated less virus than control plants. Transgenic tomato plants showed a drastic reduction in symptoms and no viral accumulation at 14 days post‐inoculation. Subcellular localization of TCTP was determined in healthy and systemically infected N. benthamiana leaves. TCTP was observed in both the nuclei and cytoplasm of non‐infected cells, but only in the cytoplasm of infected cells. Our results indicate that TCTP is a growth regulator necessary for successful PepYMV infection and that its localization is altered by the virus, probably to favour the establishment of virus infection. A network with putative interactions that may occur between TCTP and Arabidopsis thaliana proteins was built. This network brings together experimental data of interactions that occur in other eukaryotes and helps us to discuss the possibilities of TCTP involvement in viral infection.     

Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids

Lipoxygenase activity was characterized in the gill tissue of fresh-water trout. Incubation of arachidonic acid with gill preparations yielded 12-hydroxyeicosatetraenoic acid as the major product, suggesting a 12-lipoxygenase. Eicosapentaenoic acid was similarly converted to the 12-hydroxyeicosapentaenoic acid. Both arachidonic acid and docosahexaenoic acid were converted with equal apparent velocities and affinities into single monohydroxy derivatives. Analyses of the hydroxy product of docosahexaenoic acid were consistent with 14-hydroxydocosahexaenoic acid. This enzyme activity was localized to the cytosolic fraction and displayed a broad pH optimum around pH 7. The enzyme was insensitive to the cyclooxygenase inhibitors indomethacin and aspirin but activity was strongly inhibited in the presence of the lipoxygenase inhibitors, SnCl2 (5 mM), esculetin (10 microM) and eicosatetraynoic acid (100 microM).     

A GC cluster repeat is a hotspot for mit- macro-deletions in yeast mitochondrial DNA.

Summary In a random collection of mit^– mutations of the yeast strain 777-3A we find that deletions are exceptionally frequent in the OXI3 gene, a large mosaic gene coding for subunit I of cytochrome oxidase. About 10% of all oxi3 ^– mutants carry the same macro-deletion, del-A, extending from the 5 non-translated leader of OXI3 to intron 5b of this gene. Determination of the respective wild-type sequences and of the del-A junction sequence revealed that the end-points of the deletion are in two GC clusters with 31 by sequence identity which are located at a distance of 11.3 kb. We speculate that not only the sequence identity of the two GC clusters but also the palindromic structure of these putatively mobile elements of yeast mitochondrial DNA (mtDNA) plays a role in deletion formation.