Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Zur Dynamik feiner Pseudopodien von Hochmoor-Amöben

Zusammenfassung Es werden verschiedene Vorgänge an sehr zarten Pseudopodien beschrieben und mit Hilfe der an submikroskopischen Fibrillen (schraubig gebauten Mikrofibrillen und Mikroröhrchen der Elektronenmikroskopie) auftretenden Torsionsspannungen und Rotationen gedeutet.

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On the dynamics of slender pseudopodia of bog amebae

Summary Different processes which occur on very slender pseudopods are described. They are interpreted with the help of torsional forces and revolutions of the submicroscopic fibrils (helical microfibrils and microtubules in electron microscopy).

     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Synchronisation der circadianen Aktivitätsperiodik von Eidechsen durch Temperaturcyclen verschiedener Amplitude

Summary The locomotor activity of lizards was recorded in constant light, and temperature cycles of different amplitude (Fig. 1). It was determined whether or not the circadian periodicity of the animals was entrained by the external cycles. With diminishing amplitude of the temperature cycle, the number of animals entrained decreased (table); but even with daily temperature fluctuations as small as 0.9° C, 2 out of 8 animals were still entrained.In some cases, an influence of the cyclically fluctuating ambient temperature was noticeable though the circadian rhythm was not fully entrained: it showed relative coordination with the external cycle (Fig. 2).The high sensitivity to ambient temperature fluctuations demonstrated here, might account for some of the findings where the circadian period, in constant conditions, did not deviate from that under natural conditions. Some cases in which the circadian period returned to the natural phase in constant conditions following a phase shift, might have a similar cause.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

A “Bloom” in the Water of Amursky Bay (Sea of Japan) Caused by the Dinoflagellate Oxyrrhis marina Dujardin, 1841

A bloom in the water of Amursky Bay (Sea of Japan) caused by the dinoflagellate Oxyrrhis marina Dujardin, 1841 was recorded for the first time. The highest density of this species in the bloom area was 443.3 million cells/liter. The abundant development of microalga was observed from July to September 2002 at a water temperature of 17–24.5°C and a salinity of 7–18. Changes in the density of O. marina and other species of phytoplankton during the bloom period are analyzed. Possible reasons for the blooms of O. marina in Amursky Bay are discussed.     

A potassium conductance activated by hyperpolarization in paramecium

Summary Voltage clamp studies show that the wild-type membrane ofParamecium tetraurelia contains a conductance component which is sensitive to hyperpolarization. This component manifests itself as anomalous, or inward going, rectification of membrane voltage in response to applied constant current pulses and as a hyperpolarizing spike when no K is added to the external solution (Y. Satow, C. Kung, 1977.J. Comp. Physiol. 11999). Like the conductances which underlie anomalous rectification in other cells, the hyperpolarization-sensitive conductance inParamecium is specific for K, and the magnitude of the voltage-dependent conductance change depends not only on voltage but also on external potassium concentration. The internal potassium ion concentration ofParamecium is calculated to be between 17 and 18mm.     

DNA homology and adsorption specificity of Pseudomonas aeruginosa virulent bacteriophages

Summary The DNA homology and adsorption specificity of newly isolated virulent bacteriophages of P. aeruginosa have been studied. On the basis of this analysis all phages were divided into four groups: k, m, mnP78-like and mnF82-like bacteriophages. DNA's of k as well as m phages were shown to possess different restriction patterns although they have an extensive homology. Unlike other groups, k phages were characterized by the presence of T4 DNA ligase-repaired, single-chain breaks.Abbreviations kbp kilobase pairs - EM electron microscopy     

The rapid switch-off of nitrogenase activity in obligate methane-oxidizing bacteria

Nitrogenase activity in the obligate methaneoxidizing bacterium Methylococcus capsulatus (Bath) was added ammonia. This observation was extended to include other ammonia. This observation was extended to include other representative N₂-fixing species of methanotrophs. The ammonia switch-off of nitrogenase in M. capsulatus (Bath) was reversed on washing cells to remove excess ammonia, in the presence of chloramphenicol, suggesting that a form of covalent modification of nitrogenase may occur. Replacing the oxidizable substrate methanol with formaldehyde, formate, ethanol or hydrogen had no effect on nitrogenase switch-off. A number of potential nitrogen sources or intermediates of nitrogen metabolism such as glutamine, asparagine, glutamate and alanine when tested, did not effect switch-off. However, the rapid inhibition of nitrogenase activity of M. capsulatus (Bath) could be achieved by adding the uncoupler carbonylcyanide m-chlorophenylhydrazone or nitrite. The glutamine synthetase inhibitor methionine sulphoximine blocked the switch-off effect of ammonia, indicating that the metabolism of ammonia may be essential for switch-off to occur. Inhibitors of glutamate synthase did not alleviate the ammonia switch-off response. Methionine sulphoximine did not alleviate the rapid inhibition of nitrogenase by carbonylcyanide m-chlorophenylhydrazone indicating that the shortterm regulation of nitrogenase by uncouplers and ammonia proceed via different mechanisms.Abbreviations MSX methionine-DL-sulphoximine - DON 6-diazo-5-oxo-L-norleucine - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase (glutamate synthase) - CCCP carbonylcyanide m-chlorophenyl hydrazone     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

The suppression of Haemoglobin E synthesis

The blood of two infants with Haemoglobin E trait and a form of -thalassaemia (Haemoglobin H Disease) was examined and it was confirmed that the proportion of Haemoglobin A:E was higher than in uncomplicated Haemoglobin E trait.Haemoglobin H ( ₄ ^A ) was added to the haemoglobin solution from a Haemoglobin E trait carrier. This mixture was dissociated into its ₂, ₂ ^A and ₂ ^E subunits, and these were then recombined. The proportion of A:E had risen to that found in vivo in Haemoglobin E trait carriers with Haemoglobin H Disease.It is suggested that competition between ^A and ^E for -chains may be an example of the mechanism by which -thalassaemia interacts with -chain abnormal haemoglobins.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Rethinking neurasthenia: The illness concepts of Shenjing Shuairuo among Chinese undergraduates in Hong Kong

Neurasthenia is both a Western disease construct and a popular Chinese illness concept (shenjing shuairuo, SJSR). Using a self-report questionnaire, we examined 148 Hong Kong Chinese undergraduates' concept of its epidemiology, symptomatology, etiology and treatment. Notwithstanding that fatigue is thesine qua non of neurasthenia in Western nosology, subjects believed that SJSR was compatible with a diversity of symptoms which fell, on factor analysis, into the neurotic, psychotic, somatic and dysfunctional subgroups. Contrary to the popular portrayal of SJSR as a physical or chronic fatigue disorder, the most common perceived symptoms were anxiety, insomnia, depression and fright. Logically, psychological etiology and remedy were highly emphasized. The perceived high prevalence, non-aggressive nature and symptomatic diversity of SJSR attested to the notion that it might camouflage and destigmatize psychiatric labels of insanity. The contextual study of neurasthenia illustrates how in its search for legitimacy an originally Western concept adapts, transforms, and acquires distinctive local meanings in a non-Western culture.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Plastid Sequence Evolution: A New Pattern of Nucleotide Substitutions in the Cucurbitaceae

Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A=adenine, G=guanine) or pyrimidines (C=cytosine, T=thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the GACTG substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.     

An α subunit-deficient form of eukaryotic protein synthesis initiation factor eIF-2 from rabbit reticulocyte lysate and its activity in ternary complex formation

Eukaryotic protein synthesis initiation factor eIF-2 is usually isolated as a heterotrimer (). By use of Sephacryl S-300 fractionation an subunit-deficient form of eIF-2 was identified in impure preparations from rabbit reticulocyte lysate and it appeared in these preparations to be still active in formation of the ternary complex (eIF-2.GTP.Met-tRNAi). Subsequently subunit-deficient eIF-2 was further purified and this appeared to have retained ternary complex forming activity. Together with a suggested lack of involvement of the subunit this implies that the subunit was not required for activity and the subunit bound both GTP and Met-tRNA_i in formation of the ternary complex. The identification and study of subunit-deficient eIF-2 thus elucidated the involvement of the subunits in binding of GTP and Met-tRNA_i to produce the ternary complex in polypeptide chain initiation.