The effects of the piroplasm Babesia bigemina on the survival and reproduction of the blue tick,Boophilus decoloratus

Seven strains of the insect pathogenic fungus Verticillium lecanii have been examined in the Calliphora erythrocephala bioassay for the production, in surface culture on Czapek Dox medium, of insecticidal secondary metabolites. One strain which had lost its pathogenicity on storage yielded no active compounds. The remainder yielded dipicolinic acid (pyridine-2,6-dicarboxylic acid) which was responsible for the insecticidal activity of acidic extracts. Neutral extracts from two strains contained novel insecticidal C₂₅ compounds.     

Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Substrate stereochemistry of isovaleryl-CoA dehydrogenase elimination of the 2-pro-R hydrogen in biotin-deficient rats

(2R)-[³H]Isovaleric acid and (2S)-[³H]isovaleric acid (ammonium salts) have been synthesized. These substances, mixed with [1-¹⁴C]isovalerate, have been administered to biotin-deficient rats, which accumulate β-hydroxyisovaleric acid in their urine, the metabolite being formed via isovaleryl-CoA and β-methylcrotonyl-CoA. The results show that most of the tritium from (2R)-[³H]isovalerate was lost, and most of the tritium from (2S)-[³H]isovalerate retained in the conversion to β-hydroxyisovalerate. The stereochemistry of the isovaleryl-CoA dehydrogenase reaction is compared with the stereochemistry of other short-chain acyl-CoA dehydrogenase reactions.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

The moose,purine degradation,and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.     

Evolutionary loss of ascorbic acid synthesizing ability

The ability to synthesize ascorbic acid has been found only in terrestrial vertebrates. The ability is not present in certain passerine birds, in fruit-eating bats, in guinea-pigs and in Anthropoidea. We postulate that these species lost this ability by a neutral evolutionary change that occurred sporadically by mutation. The change was adopted in the genetic make-up of a few groups of birds and mammals that are widely-scattered in phylogeny. Many herbivorous vertebrate species which consume diets high in ascorbic acid have retained the ability to synthesize it, so that its loss does not appear to be adaptive.     

Chemical studies of a factor which elicits feeding behaviour in Dugesia dorotocephala

Extracts of brine shrimp have been studied and chemically fractionated in order to clarify the nature of the compounds which elicit feeding behaviour in Dugesia dorotocephala. The active principle is heat stable and has a molecular weight between 700 and 2000. Neither oxidation with hydrogen peroxide nor digestion with proteolytic enzymes destroyed the factor, although activity was lost after hydrolysis with acid. The factor has been partially purified. The available data suggest that the factor active with D. dorotocephala is larger in molecular weight, and probably chemically more complex, than those active in the coelenterates. At all stages of purification, the factor elicits the entire feeding behaviour of D. dorotocephala. No evidence was obtained for the existence of more than a single factor.     

CsCl Density Gradient Centrifugation Studies of Intact Bacterial Cells

Cells of Escherichia coli have been successfully banded in CsCl density gradients and a portion of the population reclaimed in a viable state. Differentiation between two strains of this organism in a CsCl density gradient has been demonstrated also. Several studies were undertaken to see whether differences could be detected between two samples of cells of the same strain which had been subjected to different conditions. The results were as follows: (a) Introduction of a heavy label (5-bromouracil) into the DNA during a 90 minute period did not produce an observable change in cell density. (b) Removal of a required amino acid from the growth medium of an E. coli auxotroph resulted in an increase in both the density and heterogeneity of the cells. (c) Exposure of cells to 27 kr of gamma radiation, followed by a period during which portions of both DNA and RNA were lost, yielded two distinct bands, one at the normal position in the gradient and the other shifted to a lighter region.     

Phage formation in Staphylococcus muscae cultures. XI. The synthesis of ribonucleic acid,desoxyribonucleic acid,and protein in uninfected bacteria

1. The synthesis of ribonucleic acid, desoxyribomicleic acid, and protein in S. muscae has been studied: (a) during the lag phase, (b) during the early log phase, and (c) while the cells are forming an adaptive enzyme for lactose utilization. 2. During the lag phase there may be a 60 per cent increase in ribonucleic acid and protein and a 50 per cent increase in dry weight without a change in cell count, as determined microscopically, or an increase in turbidity. 3. During this period, the increase in protein closely parallels the increase in ribonucleic acid, in contrast to desoxyribonucleic acid, which begins to be synthesized about 45 minutes after the protein and ribonucleic acid have begun to increase. 4. The RNA N/protein N ratio is proportional to the growth rate of all S. muscae strains studied. 5. While the RNA content per cell during the early log phase depends upon the growth rate, the DNA content per cell is fairly constant irrespective of the growth rate of the cell. 6. Resting cells of S. muscae have approximately the same RNA content per cell irrespective of their prospective growth rate. 7. While the cells are adapting to lactose, during which time there is little or no cellular division, there is never an increase of protein without a simultaneous increase in ribonucleic acid, the RNA N/protein N ratio during these intervals being approximately 0.15. 8. Lactose-adapting cells show a loss of ribonucleic acid. The purines-pyrimidines of the ribonucleic acid can be recovered in the cold 5 per cent trichloroacetic acid fraction, but the ribose component is completely lost from the system. 9. The significance of these results is discussed in relation to the importance of ribonucleic acid for protein synthesis.     

Some properties of Photosystem II preparations from the cyanobacterium Synechococcus sp. — presence of an l-amino acid oxidase in Photosystem II complexes from Synechococcus sp.

A highly active O₂-evolving Photosystem II complex has been purified from the cyanobacterium Synechococcus sp., and this complex has been compared with the Photosystem II complex previously isolated from this cyanobacterium (Ohno, T., Satoh, K. and Katoh, S. (1986) Biochim. Biophys. Acta 852, 1–8). Further treatment of the O₂-evolving complex with the detergent sodium taurodesoxycholate resulted in a complex which consisted mainly of the 47 and 40 kDa peptides and which had lost the O₂-evolving activity, but which could still reduce 2,6-dichlorophenolindophenol with 1,5-diphenylcarbazide. Previously, we have shown that a flavoprotein of 49 kDa which has an l-amino acid oxidase activity under certain conditions, is a component of highly active Photosystem II preparations from the cyanobacterium Anacystis nidulans (Pistorius, E.K. and Gau, A.E. (1986) FEBS Lett. 206, 243–248). Based on immunological studies with the antiserum raised against the l-amino acid oxidase protein from A. nidulans, we show that a protein which cross-reacts with this antiserum is present in the highly purified Photosystem II preparations from Synechococcus sp. Moreover, an l-amino acid oxidase activity could also be detected in Photosystem II preparations from Synechococcus sp. The enzyme preferentially oxidizes basic l-amino acids as l-arginine, l-ornithine, 2,3-diamino propionic acid and l-citrulline. In contrast to the enzyme from A. nidulansl-lysine is not oxidized. The here shown presence of an l-amino acid oxidase protein in Photosystem II preparations from Synechococcus sp. is an additional support of our hypothesis that a flavoprotein is a functional component of the water-oxidizing enzyme complex.     

Biogenesis of mitochondria 20 the effects of altered membrane lipid composition on mitochondrial oxidative phosphorylation inSaccharomyces cerevisiae

1. The lipid composition of mitochondria isolated from a fatty acid desaturase mutant ofSaccharomyces cerevisiae may be extensively manipulated by growing the organism on defined supplements of unsaturated fatty acid (UFA).
2. The fatty acid composition of the mitochondrial lipids closely follows that of the whole cells from which the mitochondria are isolated. UFA-depleted mitochondria contain normal levels of sterols, neutral lipids and total phospholipids, but have much lower levels of phosphatidyl inositides.
3. UFA-depleted mitochondria possess a full complement of cytochromes, oxidase both NAD-linked and flavoprotein-linked substrates at normal rates, and have levels of succinate and malate dehydrogenases similar to those of UFA-supplemented mitochondria. However, UFA-depletion has a marked effect on the ability of cytochromec to reactivate the NADH oxidase activity of cytochromec-depleted mitochondria.
4. The efficiency of oxidative phosphorylation decreases progressively with the UFA content of the mitochondria, and oxidative phosphorylation is completely lost in mitochondria containing approximately 20% UFA.
5. The incorporation of UFA into the lipids of UFA-depleted mitochondriain vivo results in a recoupling of oxidative phosphorylation. Recoupling is insensitive to both chloramphenicol and cycloheximide, indicating that all the proteins necessary for oxidative phosphorylation are present in UFA-depleted mitochondria, and that the less of oxidative phosphorylation is a purely lipid lesion.
6. ATPase activity is apparently unaffected by UFA-depletion, but³²P_i-ATP exchange activity is lost in mitochondria which have been extensively depleted in UFA.
7. Valinomycin stimulates the respiration of UFA-supplemented mitochondria in media containing potassium, but has no effect on the respiration of UFA-depleted mitochondria, suggesting that active transport of potassium is lost as a result of UFA-depletion.

     

Relaxation of selective constraints causes independent selenoprotein extinction in insect genomes

Background

Selenoproteins are a diverse family of proteins notable for the presence of the 21st amino acid, selenocysteine. Until very recently, all metazoan genomes investigated encoded selenoproteins, and these proteins had therefore been believed to be essential for animal life. Challenging this assumption, recent comparative analyses of insect genomes have revealed that some insect genomes appear to have lost selenoprotein genes.

Methodology/Principal Findings

In this paper we investigate in detail the fate of selenoproteins, and that of selenoprotein factors, in all available arthropod genomes. We use a variety of in silico comparative genomics approaches to look for known selenoprotein genes and factors involved in selenoprotein biosynthesis. We have found that five insect species have completely lost the ability to encode selenoproteins and that selenoprotein loss in these species, although so far confined to the Endopterygota infraclass, cannot be attributed to a single evolutionary event, but rather to multiple, independent events. Loss of selenoproteins and selenoprotein factors is usually coupled to the deletion of the entire no-longer functional genomic region, rather than to sequence degradation and consequent pseudogenisation. Such dynamics of gene extinction are consistent with the high rate of genome rearrangements observed in Drosophila. We have also found that, while many selenoprotein factors are concomitantly lost with the selenoproteins, others are present and conserved in all investigated genomes, irrespective of whether they code for selenoproteins or not, suggesting that they are involved in additional, non-selenoprotein related functions.

Conclusions/Significance

Selenoproteins have been independently lost in several insect species, possibly as a consequence of the relaxation in insects of the selective constraints acting across metazoans to maintain selenoproteins. The dispensability of selenoproteins in insects may be related to the fundamental differences in antioxidant defense between these animals and other metazoans.     

Properties of Rhizopus stolonifer Polygalacturonase, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings

Some properties of the polygalacturonase-elicitor from the filtrates of Rhizopus stolonifer cultures have been examined in an attempt to understand its mode of action as an elicitor of casbene synthetase activity in castor bean seedlings. Both the polygalacturonase activity and the elicitor activity are heat-labile with similar heat-sensitivity profiles. Also, the catalytic activity of the enzyme is lost on treatment with sodium periodate, as had been shown previously for the elicitor activity. The pH optimum of the enzyme activity with polygalacturonic acid as the substrate is 4.9. Exposures of germinating castor bean seedlings to the elicitor for short-term periods of 1 to 10 minutes followed by washing and incubation in sterile, distilled water are partially effective in elicitation in comparison with the continuous exposure of the seedlings over 11 hours to the same amount of the elicitor. The initial rate of reaction catalyzed by the enzyme is about 3 times faster with polygalacturonic acid as a substrate than with partially (50%) methylated polygalacturonic acid (pectin). The K_m value of the enzyme for polygalacturonic acid is about 4.2 millimolar in terms of monomeric units and about 0.07 millimolar in terms of polymer concentration. Examination of the types of products formed by the action of the enzyme suggests that it is an endo-hydrolase. The amino acid composition of this enzyme is similar to those of other extracellular fungal proteins reported. The carbohydrate moiety of the glycoprotein polygalacturonase-elicitor is composed of 92% mannose and 8% glucosamine by gas chromatography-mass spectrometry analysis. The linkage group analysis of the carbohydrate moiety showed that mannosyl residues which are 1,2-linked comprise about 70% of the total glycosyl residues and demonstrated the presence of some 1,3,6- and 1,2,6-linked branching mannosyl residues.     

Identification and functional characterization of polyunsaturated fatty acid elongase (McELOVL5) gene from pike eel (Muraenesox cinereus)

The cDNA coding for a polyunsaturated fatty acid elongase (McELOVL5) was isolated from the brain of the pike eel (Muraenesox cinereus) being based on available sequences in 23 types of fish. Four sequence variants were identified with different amino acid substitutions as compared with two clones of McELOVL5 gene (McELOVL5 11.7 and McELOVL5 12.4). When the two variants of McELOVL5 were expressed in Saccharomyces cerevisiae, the two recombinant yeasts elongated γ-linolenic acid (GLA, 18:3n-6) to di-homo-γ-linolenic acid (DGLA, 20:3n-6) but differed in the rate of GLA conversion to DGLA. Cells transformed with McELOVL5 12.4 also converted arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3) to docosatetraenoic acid (22:4n-6) and docosapentaenoic acid (22:5n-3), respectively. However McELOVL5 11.7 lost its function for the elongation of C₂₀ fatty acids. The four sequence variants have changed substrate specificities. Three-dimensional models of the McELOVL5 proteins are suggested.     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

Mutational Tuning of Galectin-3 Specificity and Biological Function

Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1–3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.     

Expression and evolution of hexamerins from the tobacco hornworm,Manduca sexta,and other Lepidoptera

Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins.     

Acetic Acid Increases Stability of Silage under Aerobic Conditions

The effects of various compounds on the aerobic stability of silages were evaluated. It has been observed that inoculation of whole-crop maize with homofermentative lactic acid bacteria leads to silages which have low stability against aerobic deterioration, while inoculation with heterofermentative lactic acid bacteria, such as Lactobacillus brevis or Lactobacillus buchneri, increases stability. Acetic acid has been proven to be the sole substance responsible for the increased aerobic stability, and this acid acts as an inhibitor of spoilage organisms. Therefore, stability increases exponentially with acetic acid concentration. Only butyric acid has a similar effect. Other compounds, like lactic acid, 1,2-propanediol, and 1-propanol, have been shown to have no effect, while fructose and mannitol reduce stability.     

The biosynthesis of dicoumarol

Micro-organisms have been isolated that can utilize o-coumaric acid as a sole carbon source with the subsequent production of 4-hydroxycoumarin and dicoumarol. One of these organisms, Penicillium jenseni, has been used to examine the biosynthesis of dicoumarol. Certain thermophilic fungi have also been found that can convert o-coumaric acid into dicoumarol.