Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.     

The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM-GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1-CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.     

The influence of aquatic surface respiration (ASR) on cardio-respiratory function of the serrasalmid fish Piaractus mesopotamicus

When exposed to severely hypoxic water, many teleosts skim the better oxygenated surface layer (aquatic surface respiration, ASR). Information is scarce concerning the thresholds triggering ASR and its cardio-respiratory consequences. To assess the ambient conditions leading to ASR and to evaluate its effects on cardio-respiratory function, we exposed specimens of Piaractus mesopotamicus to gradual hypoxia (water oxygen tension ranging from 120 to 10 torr) with or, alternatively, without access to the surface. Concurrently, ASR, cardiac and respiratory frequencies, O₂ uptake and gill ventilation were monitored. With surface access, ASR developed below the critical tension for O₂ uptake (34 torr) by normal gill ventilation. Moreover, the time spent in ASR increased with prolonged hypoxic exposure to a maximum of 95% of total time. Without surface access, the species exhibited hypoxic bradycardia, that had not occurred in the group with fully developed ASR. Even without ASR, P. mesopotamicus recovered readily from hypoxic exposure, showing that this species possesses a number of mechanisms to cope with environmental hypoxia.     

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

The cortical actin cytoskeleton beneath the plasma membrane represents a physical barrier that vaccinia virus has to overcome during its exit from an infected cell. Previous observations using overexpression and pharmacological approaches suggest that vaccinia enhances its release by modulating the cortical actin cytoskeleton by inhibiting RhoA signalling using the viral protein F11. We have now examined the role of F11 and its ability to interact with RhoA to inhibit its downstream signalling in the spread of vaccinia infection both in vitro and in vivo. Live cell imaging over 48 hours reveals that loss of F11 or its ability to bind RhoA dramatically reduces the rate of cell-to-cell spread of the virus in a cell monolayer. Cells infected with the ΔF11L virus also maintained their cell-to-cell contacts, and did not undergo virus-induced motility as observed during wild-type infections. The ΔF11L virus is also attenuated in intranasal mouse models of infection, as it is impaired in its ability to spread from the initial sites of infection to the lungs and spleen. Loss of the ability of F11 to bind RhoA also reduces viral spread in vivo. Our results clearly establish that viral-mediated inibition of RhoA signalling can enhance the spread of infection not only in cell monolayers, but also in vivo.     

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.     

Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in <Emphasis Type="Italic">Acca sellowiana</Emphasis> (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.     

Phosphorylation of histone H3S10 in animal chromosomes: is there a uniform pattern?

Phosphorylation of serine 10 in histone H3 (H3S10ph) has been extensively analyzed and appears to be a conserved chromatin change associated with chromosome condensation in different eukaryotic organisms. In this work, we report the distribution of H3S10ph during meiosis in monocentric and holokinetic chromosomes of 6 insect species and in mitotic chromosomes of 7 mammalian species, aiming to investigate the labeling patterns in phylogenetically distant groups. The results indicated a very similar phosphorylation timing and distribution pattern among insects. The sex chromosomes of insects analyzed were always undercondensed and hypophosphorylated. Similarly, the micro chromosomes of the bug Pachylis aff pharaonis were also undercondensed and hypophosphorylated. Holokinetic chromosomes of bugs and monocentric chromosomes of grasshoppers and beetles displayed identical phosphorylation pattern in spite of the difference in the centromere type. Among mammals, a uniform chromosome phosphorylation was observed in marsupials, whereas bat chromosomes displayed a longitudinal banding pattern. These data indicate that, in general, the intensity of H3S10 phosphorylation in animal chromosomes is variable among the distinct chromosome types and associated with the degree of chromatin condensation at metaphase, but it may vary between different groups of animals.     

Cephalopod paralarvae and upwelling conditions off Galician waters (NW Spain)

A total of 103 cephalopod paralarvae were sampled during June 1995 in Galician waters (NW Spain). Samples were taken with Bongo nets of 300 and 500 m mesh size at 48 sampling stations along 10 transverse transects ranging from 80 to 600 m water depth. Paralarvae of loliginid squid were most abundant (40%). The Rhynchoteuthion paralarvae of ommastrephid squid accounted for 25%, whereas sepiolids comprised 23% of the total sample. Octopods were scarce, at only 6.6%. Other cephalopod families accounted for 5%. Sizes of paralarvae ranged from 1.0 to 7.1 mm mantle length. Temperature and salinity distribution showed the presence of an intense upwelling during the survey period. The sampling data obtained before and during the presence of upwelled water off Rias of Pontevedra and Vigo (southern zone) showed that paralarval cephalopod abundance and distribution were closely related to the upwelled Eastern North-Atlantic Central Water (ENACW).      

Is tensegrity a unifying concept of protein folds?

We suggest that the three-dimensional architecture of globular proteins can be described in terms of tensegrets, i.e. structural elements that are held together through attractive and repulsive forces. Hard elements of tensegrets are represented by secondary structure elements, i.e. alpha-helices and beta-strands, while the role of elastic elements is played by attractive and repulsive atomic forces. Characteristics of tensegrets is that they can auto-assemble and that they respond to changes of tension in some part of the entire object through a deformation in another part, thus partially preserving their structure, despite their deformation. This latter property well explains both the folding process and the behavior of globular proteins under mild denaturing conditions, as revealed by the molten globule state.