Gene expression in autumn leaves

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.     

Insertional inactivation of the methionine s-methyltransferase gene eliminates the s-methylmethionine cycle and increases the methylation ratio

Methionine (Met) S-methyltransferase (MMT) catalyzes the synthesis of S-methyl-Met (SMM) from Met and S-adenosyl-Met (Ado-Met). SMM can be reconverted to Met by donating a methyl group to homocysteine (homo-Cys), and concurrent operation of this reaction and that mediated by MMT sets up the SMM cycle. SMM has been hypothesized to be essential as a methyl donor or as a transport form of sulfur, and the SMM cycle has been hypothesized to guard against depletion of the free Met pool by excess Ado-Met synthesis or to regulate Ado-Met level and hence the Ado-Met to S-adenosylhomo-Cys ratio (the methylation ratio). To test these hypotheses, we isolated insertional mmt mutants of Arabidopsis and maize (Zea mays). Both mutants lacked the capacity to produce SMM and thus had no SMM cycle. They nevertheless grew and reproduced normally, and the seeds of the Arabidopsis mutant had normal sulfur contents. These findings rule out an indispensable role for SMM as a methyl donor or in sulfur transport. The Arabidopsis mutant had significantly higher Ado-Met and lower S-adenosylhomo-Cys levels than the wild type and consequently had a higher methylation ratio (13.8 versus 9.5). Free Met and thiol pools were unaltered in this mutant, although there were moderate decreases (of 30%-60%) in free serine, threonine, proline, and other amino acids. These data indicate that the SMM cycle contributes to regulation of Ado-Met levels rather than preventing depletion of free Met.     

Expression and localization of human lysozyme in the endosperm of transgenic rice

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.     

A comparison of the long-term morbidity following deep circumflex iliac and fibula free flaps for reconstruction following head and neck cancer

Composite free tissue transfer has an established role in head and neck oncology for the reconstruction of the bony defect following tumor ablation, and while donor-site morbidity is variably reported, there is little consensus on the most favorable donor site. The fibula and deep circumflex iliac artery have distinct advantages in terms of the volume and length of bone in mandibular reconstruction. Few studies have compared their donor-site morbidity. The aim of this study was to compare the fibula and deep circumflex iliac artery flaps using a review of the case notes and cross-sectional review of patients attending a research clinic for validated orthopedic examination and completion of health-related quality-of-life questionnaires. Between February of 1993 and May of 2001, 44 fibula free flaps and 73 deep circumflex iliac artery free flaps were performed. Ninety-nine case notes and 36 patients were available for review of donor-site morbidity. Sixteen patients with fibula flaps and 20 patients with deep circumflex iliac artery flaps took part in the clinical examination component of the study, which was composed of a clinical examination by an orthopedic surgeon using the American Orthopedic Foot and Ankle Society ankle scoring system and the Harris hip scoring system, and two patient-completed questionnaires, the University of Washington Questionnaire and the Hospital Anxiety and Depression Scale. Subjective and objective markers of morbidity related to both flaps were similar in most parameters. However, fibula flaps were associated with more problems with donor-site healing, reduced power, and sensation. Poor orthopedic scores for both flaps were associated with notably poor scores on the University of Washington Questionnaire and the Hospital Anxiety and Depression Scale. The study would suggest that both deep circumflex iliac artery and fibula donor sites result in an acceptable and comparable morbidity for most patients, but in cases in which significant donor-site morbidity is encountered, health-related quality of life is significantly compromised.     

Two very unusual macrocyclic flavonoids from the water lily Nymphaea lotus

Three novel flavonols, myricetin-3'-O-(6"-p-coumaroyl)glucoside and two epimeric macrocyclic derivatives, as well as the known myricetin-3-O-rhamnoside and pentagalloyl glucose, have been isolated from the wild water lily Nymphaea lotus L. and identified using 2D NMR. This is the first report of such a macrocycle from any source.     

Linked redox precipitation of sulfur and selenium under anaerobic conditions by sulfate-reducing bacterial biofilms

A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixed biofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200 micro M selenite from solution during growth on lactate and sulfate. Elemental selenium and elemental sulfur were precipitated outside SRB cells. Precipitation occurred by an abiotic reaction with bacterially generated sulfide. This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can take place under low redox conditions and in the dark. The reaction represents a new means for the deposition of elemental sulfur by SRB under such conditions. A combination of transmission electron microscopy, environmental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used to reveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium in relation to biofilm elements. When pregrown SRB biofilms were exposed to a selenite-containing medium, nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix. Selenite was therefore shown to pass through the biofilm matrix before reacting with bacterially generated sulfide. This constitutes an efficient method for the removal of toxic concentrations of selenite from solution. Implications for environmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potential action of SRB in selenium transformations is presented.     

Anthocyanic vacuolar inclusions (AVIs) selectively bind acylated anthocyanins in Vitis vinifera L. (grapevine) suspension culture

Anthocyanic vacuolar inclusions (AVIs) appear as dark red-to-purple spheres of various sizes in vacuoles of grapevine (Vitis vinifera L.) cell suspension culture due to their interaction with anthocyanins. AVIs were purified and the bound anthocyanins extracted and analysed by HPLC from two lines of V. vinifera isolated from the same callus accumulating anthocyanin in the dark, yet varying in their anthocyanin profiles and accumulation. An intermediate-pigmented line (FU-1) with a 1.3:1 ratio of acylated:non-acylated anthocyanins, a colour value of 0.84 units and cyanidin and peonidin as the dominant species was compared with a high-pigmented line (FU-2) with a 1.2:1 ratio of acylated:non-acylated anthocyanins, a colour value of 3.72 units and malvidin predominating. The profile of AVI-bound anthocyanins showed an increase in acylated anthocyanins in both lines of approx. 28–29%, with no apparent preference for anthocyanin species. This resulted in a ratio of acylated:non-acylated anthocyanins of 6.2:1 for FU-1 and 4.9:1 for FU-2. The reasons for the selectivity of the AVIs for acylated (specifically p-coumaroylated) species compared with the whole cell profile are discussed.     

Transition metal complexes of buparvaquone as potent new antimalarial agents. 1. Synthesis,X-ray crystal-structures,electrochemistry and antimalarial activity against Plasmodium falciparum

New Cu(II), Ni(II), Co(II), Fe(II), and Mn(II) metal complexes of buparvaquone [3-trans(4-tert.-butylcyclohexyl)methyl-2-hydroxy-1,4-naphthoquione] (L1H) have been synthesized and characterized using IR, electron paramagnetic resonance (EPR) spectroscopy, microanalytical methods and single crystal X-ray diffraction methods. The single crystal structures were determined for ligand L1H [space group P-1 with a=6.2072(14) A, b=10.379 (2) A, c=13.840 (3) A, V=878.7(3) A(3), Z=2, D(calcd.)=1.234 mg/m(3)] and copper complex [Cu(L1)(2)(C(2)H(5)OH)(2)] C1 [space group I2/a with a=17.149(14) A, b=9.4492(8) A, c=26.946(3) A, V=4335.3(7)A(3), Z=4, D(calcd.)=1.233 mg/m(3)]. All the metal complexes along with the parent ligand have been studied for their electrochemical properties using cyclic voltammetric techniques. The compounds were tested for their in vitro antimalarial activity against Plasmodium falciparum strains. A correlation between the antimalarial activity and the redox property of these complexes is presented. The copper complex C1 exhibits significantly higher growth inhibitory activity both in vitro and in vivo than the parent ligand.     

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.