Biochemical investigation of two responses involved in the feeding behaviour of Acanthaster planci (L.). III. Food preferences

The arm-withdrawal and settlement responses associated with feeding in Acanthaster planet (L.) are elicited at differing intensities by various species of coral and by extracts of these corals. There is no clear correlation between the strengths of the two responses. The preferences shown by A. planci for certain species of whole coral depend on the previous dietary experience of the starfish, and repeated presentation of an unfamiliar coral may increase its acceptability as food.     

Biochemical stimuli for feeding in Acanthaster planci (L.)

The typical feeding response of the Crown of Thorns Starfish Acanthaster planci (L.) can be induced by mucoproteins of molecular weight greater than 200,000 daltons found in mucus obtained from corals of the genera Acropora and Fungia.     

Biochemical investigation of two responses involved in the feeding behaviour of Acanthaster planci (L). I. Assay methods and preliminary results

Material obtained by simple biochemical fractionation of aqueous extracts of coral has been tested for its ability to induce responses normally associated with feeding on live corals in the crown-of-thorns starfish, Acanthaster planci (L.) With the use of a new assay method, it has been shown that both dialysable and non-dialysable components of aqueous coral extract cause cessation of movement, settlement, and eversion of the stomach as if over a whole coral. A second response, apparently of avoidance, seen as a starfish approaches a coral prior to feeding, has also been examined; it is evoked by the dialysable fraction of coral extract.     

Saponins in eggs and larvae of A canthaster planci (L.) (Asteroidea) as chemical defences against planktivorous fish

The suggestion that saponins in eggs and larvae of Acanthaster planci (L.) serve as chemical defences was tested by feeding groups of planktivorous pomacentrid fish with random series of gelatin food particles, some with and some without crude saponin extract from A. planci. The four fish species discriminated at statistically significant levels against food particles with crude saponin extract at 1× 10^?3 and 1× 10^?5 parts of wet weight. Three species also discriminated at significant levels against particles with 1× 10^?7 parts crude saponin extract per wet weight. Degree of discrimination was strongly influenced by the state of hunger of the fish. Tastiness of particles containing saponins also influenced acceptability. The lower two concentrations of saponins used in these feeding trials were respectively two and four magnitudes less than in A. planci eggs and larvae. Thus, the saponins in eggs and larvae of A. planci are at levels detectable by, and unpalatable to, planktivorous fish and they account, at least in part, for the observed rejection of these early developmental stages by planktivorous fish.     

Seasonal changes in the activity of certain enzymes in crude tissue extracts of two common cirripedes, Balanus balanoides (L.) and B. balanus (L.).

The activity of glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (ICDH), and aldolase in tissue extracts of Balanus balanoides (L.) and B. balanus (L.) have been determined at regular intervals over a year, and an attempt made to relate the results to known biochemical changes in the animals.The work was ecologically orientated. Preliminary studies on the effects of extractant, dialysis, and possible endogenous interference were made and the subsequent procedure rigorously standardized.Transamination was investigated by TLC. LDH and aldolase show striking seasonal changes; an abundance of the former is associated with the production of semen. MDH, concerned in carbohydrate and lipid metabolism, was present in considerable quantities but there were no marked seasonal changes. GPT activity is high when reproductive products are being produced from reserves of food and the activity is also related to the production of semen. GOT showed no clear seasonal trends.The results are discussed relative to the metabolism of crustaceans: the part played by proteins and lipids is stressed.     

Isolation and characterization of two cDNAs encoding translation initiation factor 1A from rice(Oryza sativa L.).

The eukaryotic initiation factor 1A(eIF1A) is essential for transferring of the initiator Met-tRNA to 40S ribosomal subunits to form the 40S pre-initiation complex. In present study, we describe the cloning and characterization of two eIF1A genes from rice, which were designated as Oryza sativa eukaryotic initiation factor 1A genes OseIF1A-1, OseIF1A-2, respectively. Both rice elF1As shared high identities in amino acids with eIF1A proteins from other eukaryotes. The mRNA expression analysis revealed that OseIF1A-2 mRNA was much more accumulated than OseIF1A-1 in all tissues but each gene is expressed in root, stem, leaf and flowering spike in high and nearly equal level, and in immature spike in lower level. These results, together with their different location in unrooted phylogenetic tree inferred from amino acid sequences of all known eIF1As, suggested that there are two types of eIF1A genes with different function or different regulation in rice.     

Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078

The genome of Corynebacterium glutamicum NCHU 87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His₆-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414 mU mg⁻¹ protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3 Å resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70–73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.     

Isolation and characterization of mistletoe extracts (Viscum album L.). II. Effect of agglutinating and cytotoxic fractions on mouse ascites tumor cells

Lectin from mistletoe (Viscum album L.) was studied for its relations with the toxins from Viscum album, ascites tumor cells of mouse, and human immunoglobulins. Using affinity chromatography on glutaraldehyde-crosslinked IgG (human) from viscum crude extract, a fraction was isolated which exhibited full agglutination capacity and high toxicity. The supernatant showed no agglutination capacity but a strong toxic effect on mouse ascites tumor cells. This toxic effect could not be influenced by further additions of insolubilized IgG. Chromatography on DEAE cellulose also gave agglutinating fractions with toxic effects and a non-agglutinating toxic portion. Column chromatography on Sephadex G 75 allowed separation of toxic from agglutinating components. The molecular weight of the toxin remaining after lectin removal was above 10,000. Lectin was found to bind more readily to mouse ascites tumor cells than to erythrocytes.     

Studies on phytohemagglutinins. XXV. Isolation and characterization of hemagglutinins of the spindle tree seeds (Evonymus europaea L.).

A mixture of isophytohemagglutinins has been isolated from the fleshy arils of the spindle tree seeds (Evonymus europaea L.) by fractional precipitation of the saline extract of the arils by (NH4)2SO4 at a 0.40% saturation. Successive preparative disc electrophoresis on polyacrylamide gel affords separation of one slower moving component, phytohemagglutinin I, from the mixture of other isophytohemagglutinins that have a very similar electrophoretic mobility. Phytohemagglutinin I has a sedimentation coefficient Sw,20 of 7.1 S and an approximate mol. wt of 127 000. Amino acid analysis shows a high amount of aspartic acid, alanine and glycine but also significant amounts of serine, threonine, cysteine and methionine. Aspartic acid is the only N-terminal amino acid found by the dansylation technique. Phytohemagglutinin I contains glucosamine and 4.7% neutral sugar. Its approximate pI in citrate/phosphate buffer is 4.4-4.5. The metal content amounts to 0.250% Ca, 0.019% Mg, 0.034% Zn and 0.026% Cu. Mn is not present. Ultracentrifugation analysis reveals homogeneity in the sedimentation behavior of the mixture of isophytohemagglutinin, an Sw,20 of 7.1 S and an approximate mol. wt of 119 000. The mixture has an amino acid composition closely resembling that of phytohemagglutinin I and an identical pI but contains only 1.9% neutral sugar. Two N-terminal amino acids were shown to be present, aspartic acid and tyrosine. With the exception of Cu which is absent, the metal content is almost the same as that of phytohemagglutinin I. Both phytohemagglutinin I and the mixture are devoid of anti-A1 activity and show detectable anti-H, anti-B and anti-A2 erythroagglutinating activity in approximate limit concentrations of 2.5, 5 and 10 mug/ml, respectively. This activity is not influenced by the presence of EDTA, Ca2+ or Mg2+, but is stimulated by Zn2+. Mn2+ and Co2+ have an inhibitory effect. None of the simple sugars tested inhibited the hemagglutination reactions.     

Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.).

Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.     

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5′ and 3′ UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.     

Isolation and characterization of two outer membrane preparations from Escherichia coli

A method was developed for releasing specifically a part of outer membrane during spheroplast formation. A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation. The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation.Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs. Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content. It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.     

Isolation and characterization of a lectin gene from seeds of chickpea (Cicer arietinum L.).

A cDNA library was constructed in lambda TriplEx2 vector using poly (A(+)) RNA from immature seeds of Cicer arietinum. The lectin gene was isolated from seeds of chickpea through library screening and RACE-PCR. The full-length cDNA of Chichpea seed lectin(CpGL)is 972 bp and contains a 807 bp open reading frame encoding a 268 amino acid protein. Analysis shows that CpSL gene has strong homology with other legume lectin genes. Phylogenetic analysis showed the existence of two main clusters and clearly indicated that CpSL belonged to mannose-specific family of lectins. RT-PCR revealed that CAA gene expressed constitutively in various plant tissues including flower, leaf, root and stem. When chickpea lectin mRNA level was checked in developing seeds, it was higher in 10 DAF seeds and decreased throughout seed development.     

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.     

Molecular characterization and enzymatic activity of laccases in two Pleurotus spp. with different pathogenic behaviour

Pleurotus eryngii and P. ferulae, two species belonging to the P. eryngii complex, synthesize laccases, ligninolytic enzymes that play a role in the host-pathogen interaction in the first step of infection. Ecological studies have shown that although both fungi have been recognized as saprophytes, P. eryngii weakly pathogenic when colonizing the roots and stems of Eryngium campestre, whereas P. ferulae is mostly pathogenic to Ferula communis. The paper describes the genomic organization of four putative laccase genes (lac1, lac2, lac3, and lac5-like gene; gene names were assigned on the basis of sequence homologies) of P. eryngii and P. ferulae. The mRNA expression and enzymatic activity of the laccases were analysed under culture conditions where a source of lignin (wheat bran) or lyophilized roots of E. campestre or F. communis were present. These experiments indicated that the four lac-like genes were differentially regulated in the two mushrooms. Specifically, the addition of the lyophilized roots of the respective host plant to the culture media induced an advance in the mRNA expression of the four lac-like genes and a seven-fold higher total laccase activity in P. ferulae than in P. eryngii. The results obtained are discussed in relation to the possible role of laccases in the interaction of P. eryngii and P. ferulae with their respective host.     

Isolation,characterization and molecular cloning of a leaf-specific lectin from ramsons (Allium ursinum L.).

Lectins were isolated from roots and leaves of ramsons and compared to the previously described bulb lectins. Biochemical analyses indicated that the root lectins AUAIr and AUAIIr are identical to the bulb lectins AUAI and AUAII, whereas the leaf lectin AUAL has no counterpart in the bulbs. cDNA cloning confirmed that the leaf lectin differs from the bulb lectins. Northern blot analysis further indicated that the leaf lectin is tissue-specifically expressed. Sequence comparisons revealed that the ramsons leaf lectin differs considerably from the leaf lectins of garlic, leek, onion and shallot.     

Functional characterization of two microsomal fatty acid desaturases from Jatropha curcas L.

Linoleic acid (LA, C18:2) and α-linolenic acid (ALA, C18:3) are polyunsaturated fatty acids (PUFAs) and major storage compounds in plant seed oils. Microsomal ω-6 and ω-3 fatty acid (FA) desaturases catalyze the synthesis of seed oil LA and ALA, respectively. Jatropha curcas L. seed oils contain large proportions of LA, but very little ALA. In this study, two microsomal desaturase genes, named JcFAD2 and JcFAD3, were isolated from J. curcas. Both deduced amino acid sequences possessed eight histidines shown to be essential for desaturases activity, and contained motif in the C-terminal for endoplasmic reticulum localization. Heterologous expression in Saccharomyces cerevisiae and Arabidopsis thaliana confirmed that the isolated JcFAD2 and JcFAD3 proteins could catalyze LA and ALA synthesis, respectively. The results indicate that JcFAD2 and JcFAD3 are functional in controlling PUFA contents of seed oils and could be exploited in the genetic engineering of J. curcas, and potentially other plants.     

Parvalbumins from frog skeletal muscle (Rana temporaria L.). Isolation and characterization. Structural modifications associated with calcium binding.

Two parvalbumins have been isolated from the skeletal muscle of Rana temporaria L. Amino acid composition, tryptic peptide maps, isoelectric points, calcium content and ultraviolet as well as circular dichroism spectra have been determined. Investigation on antigenic properties revealed no antigenic determinants common to both components. The two protein molecules appear to belong to far related gene lineages. They are also different from the parvalbumins found in Rana esculenta muscle. Modifications of physical parameters, associated with calcium binding and dissociation are described. While antigenicity remained essentially unchanged, conformational changes were revealed by alterations of circular dichroism spectra.