Structural analysis of the bacteriophage T3 head-to-tail connector

The connector protein of bacteriophage T3, p8, has been overexpressed in Escherichia coli. Purification of the oligomers built by several copies of p8 reveals a mixed population of dodecamers and tridecamers. The percentages of these two types of oligomers differ in every culture growth, indicating that assembly of this protein depends upon the conditions of the expression system. Those cultures that generated a majority of dodecamers allowed, after purification of the connectors, the two-dimensional crystallization of the dodecamers in a tetragonal arrangement, while the tridecamers did not form crystals. The processing and averaging of several images of frozen-hydrated crystals and their internal phase comparison shows that the crystals are arranged in a P42(1)2 space group, with cell unit dimensions of 165 x 165 A. The three-dimensional reconstruction generated with images of crystals ranging from 0 degrees to 60 degrees tilt reveals a wide domain surrounded by 12 protrusions and a narrow domain that serves to interact with the tail of the bacteriophage. A channel runs along the connector wide enough to allow the translocation of a double-stranded DNA molecule into the prohead. The general structure of the T3 connector is very similar to those obtained for other nonrelated bacteriophages and strongly suggests that the shape of this important viral structure is intimately related to its function.     

Increased aortic stiffness assessed by pulse wave velocity in apolipoprotein E-deficient mice

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 +/- 4%), PWV increased significantly (3.8 +/- 0.2 m/s) compared with controls (2.9 +/- 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 +/- 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.     

Characterization of a Rab11 homologue in Trypanosoma cruzi

Vesicle trafficking between organelles occurs through fusion of donor and specific acceptor membranes. This process is highly regulated and ensures proper direction in sorting and packaging of a number of molecules in eukaryotic cells. Monomeric GTPases of the Rab family play a pivotal role in the control of membrane fusion and vesicle traffic. In this paper, we characterize a Trypanosoma cruzi Rab 11 homologue (TcRab11) that shares at, the amino acid level, 40% similarity with human rab11, Arabdopsis thaliana rab11 and yeast rab11 homologue genes. Western blot analysis, using a polyclonal rabbit antiserum raised against a synthetic peptide derived from the COOH-terminus of predicted the TcRab11 protein, reacted to a 26kDa protein. In immunofluorescence assays, TcRab 11, was shown to be expressed in epimastigote and amastigote forms, but it was absent in trypomastigotes. Interestingly, the TcRab11 product seems to be located at the reservosome complex, a site of active endocytosis and vesicle fusion present only in the epimastigote stage. Therefore, TcRab11 may represent the first molecular marker of this peculiar organelle.     

Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases

The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P相似文献   

A limonoid from Trichilia estipulata

The limonoid 21,24,25,26,27-pentanor-15,22-oxo-7alpha,23-dihydroxy-apotirucalla(eupha)-1-en-3-one was isolated from the dichloromethane extract of the stem bark of Trichilia estipulata. Its structure was established by spectroscopic methods (UV, EIMS, 1H and 13C NMR, HMQC and HMBC).     

Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum

Given the importance of the antitumor/antiviral lignans, podophyllotoxin and 5-methoxypodophyllotoxin, as biotechnological targets, their biosynthetic pathways were investigated in Podophyllum peltatum and Linum flavum. Entry into their pathways was established to occur via dirigent mediated coupling of E-coniferyl alcohol to afford (+)-pinoresinol; the encoding gene was cloned and the recombinant protein subsequently obtained. Radiolabeled substrate studies using partially purified enzyme preparations next revealed (+)-pinoresinol was enantiospecifically converted sequentially into (+)-lariciresinol and (-)-secoisolariciresinol via the action of an NADPH-dependent bifunctional pinoresinol/lariciresinol reductase. The resulting (-)-secoisolariciresinol was enantiospecifically dehydrogenated into (-)-matairesinol, as evidenced through the conversion of both radio- and stable isotopically labeled secoisolariciresinol into matairesinol, this being catalyzed by the NAD-dependent secoisolariciresinol dehydrogenase. (-)-Matairesinol was further hydroxylated to afford 7'-hydroxymatairesinol, this being efficiently metabolized into 5-methoxypodophyllotoxin. Thus much of the overall biosynthetic pathway to podophyllotoxin has been established, that is, from the dirigent mediated coupling of E-coniferyl alcohol to the subsequent conversions leading to 7'-hydroxymatairesinol.     

Purification and characterisation of vanadium haloperoxidases from the brown alga Pelvetia canaliculata

Two enzymes characterised as iodoperoxidases (PcI and PcII), with vanadium-dependent activity, have been purified from the brown alga Pelvetia canaliculata (L.) Decne et Thur. (Fucaceae, Phaeophyceae), collected in the Northern Portuguese coast, at Viana do Castelo. The relative molecular masses were 166 kDa for PcI and 416 kDa for PcII, as determined by gel filtration. SDS-PAGE shows that PcI has just one band corresponding to a subunit of 66 kDa, while PcII shows four bands (66, 72, 157 and 280 kDa). The following kinetic parameters have been determined from a steady-state analysis of the oxidation of iodide by H2O2: PcI, pHopt = 6.0, KM(I-) = 2.1 mM, KM(H2O2) = 110 microM, Ki(I-) = 127 mM; and PcII, pHopt = 6.5, KM(I-) = 2.4 mM, KM(H2O2) = 20 microM and Ki(I-) = 69 mM. These iodoperoxidases are thermostable, as also observed for vanadium bromo- and chloroperoxidases.     

Low courtship song variation in south and southeastern Brazilian populations of Drosophila meridionalis (Diptera, Drosophilidae)

Drosophila meridionalis is a cactus-breeding species with a wide distribution in South America. Most populations of this species are geographically isolated, what provides a promising scenario for studying evolution. Former studies of this species revealed a remarkable karyotypic variation among its populations. Up to six distinct metaphase chromosomes were described, showing that this species is polymorphic at least at the chromosomal level. In order to elucidate the taxonomic status of populations showing different metaphase chromosomes, we analyzed the courtship song of five populations of D. meridionalis in South and Southeastern Brazil. In addition, we analyzed the metaphase chromosomes of each population. Our results show that, despite the two karyotype observed, most courtship song parameters did not vary among the populations. Altogether, our results suggest that D. meridionalis from South and Southeastern Brazil represents one species with an inter-population chromosomal variability.     

Worker life tables, survivorship, and longevity in colonies of Bombus (Fervidobombus) atratus (Hymenoptera: Apidae)

Survivorship curves and longevity of workers were studied in two queenright and two queenless colonies of Bombus (Fervidobombus) atratus. Survivorship curves for workers of all colonies were, in general, convex, indicating an increasing mortality rate with increasing age. The mean longevity for the workers from queenright colonies, 24.3 days and 17.6 days, was not significantly different from that in queenless colonies, 21.2 days and 20.2 days. In all colonies workers started foraging activities when aged 0-5 days, and the potential forager rates rose progressively with increasing age. Mortality rates within each age interval were significantly correlated with the foraging worker rates in all colonies. Only in two of the colonies (one queenright and one queenless) longevity was significantly correlated with worker size. The duration of brood development period seems to be one of the most important factors influencing adult worker longevity in bumble bee species.