Isolation, characterization, and linkage analyses of 74 novel microsatellites in Barramundi (Lates calcarifer).

Barramundi (Lates calcarifer) is an important marine food fish species in Southeast Asia and Australia. Seventy-four novel microsatellites were isolated from a genomic DNA library enriched for CA repeats and were characterized in 24 unrelated individuals. Among the 74 microsatellites, 71 were polymorphic, with an average allele number of 7.0 +/- 3.6/locus. The average expected heterozygosity of these polymorphic markers was 0.66. Sixty-three of the 71 polymorphic microsatellites conformed to Hardy-Weinberg equilibrium. Linkage analyses were conducted in a reference family, leading to the assignment of 34 novel microsatellites and 16 published markers in 16 linkage groups. The novel microsatellites developed in this study will contribute significantly to the construction of a first-generation linkage map for mapping of quantitative trait loci in Barramundi, and supply a large choice of markers for studies on population genetics, stock management, and pedigree reconstruction.     

Seasonal changes of root hydraulic conductance (KRL) in four forest trees: an ecological interpretation

This paper deals with the possibility of relating root hydraulic parameters to an ecological index describing the continentality/oceanicity of four forest trees. Root hydraulic conductance ($K_R$) of seedlings of Fagus sylvatica L., Quercus ilex L., Quercus suber L. and Quercus pubescens Willd. was measured in May, August and November 1996. $K_R$ was calculated in terms of the relation of the water flow through intact root systems in situ measured with the pressure chamber, and the pressure driving the flow. The sufficiency of the root system to supply the foliage was estimated by dividing $K_R$ by the seedlings leaf surface area ($A_L$) thus obtaining $K_RL$. In the spring, $K_RL$ was largest in F. sylvatica and smallest in Q. pubescens with intermediate values recorded in Q. ilex and Q. suber. All the species studied showed a large decline in $K_RL$ just prior to the winter rest except for Q. suber which mantained $K_RL$ approximately constant through the period of study. In most cases, $K_RL$ changed in accordance with analogous changes in the flow. When the total seedlings' leaf surface area ($A_L$) was plotted versus $K_RL$, it appeared that $K_RL$ of Q. pubescens increased with $A_L$, proportionally, while $K_RL$ of F. sylvatica was inversely related to $A_L$. This, together with the largest $K_RL$ recorded in the summer in Q. pubescens, was interpreted as advantageous to this species (which is adapted to semi-arid environments) in that: (a) roots could supply water to foliage efficiently even during the adverse season and (b) the foliage growth could be sustained even in summer.No statistically significant relation of $K_RL$ to the continentality index calculated for the four species studied on the basis of their European distribution, was found to exist. Nonetheless, our data appear to be encouraging for future research aimed at better interpreting the typical distribution areas of plant species.     

An Endogenous Aminoenkephalinase Inhibitor: Purification and Characterization of Arg0-Met5-Enkephalin from Bovine Striatum

Abstract: Arg⁰-Met⁵-enkephalin (Arg⁰-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio-Sil C₈, semipreparative HPLC Radial-Pak C₁₈, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C₁₈, Lichromsorb C₁₈, and μBondapak C₁₈. The peptide content was followed by radioimmunoassay with an antibody against synthetic Met-enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg⁰-MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg⁰-MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met-( R )- and Met-( S )-sulfoxide. The reduced Arg⁰-MEK inhibited aminoenkephalinase with a K _i of 2.2 µ M , and its sulfoxide analogue inhibited it with a K _i of 8.9 µ M . The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED₅₀ of 5 µ M , and the effect could be reversed by equimolar naloxone. Our data indicate that Arg⁰-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.     

Mutagenic and membranal effect of a phytotoxic molecule isolated from olive leaves parasitized by the fungusCycloconium oleaginum Cast

A phytotoxic substance (C₂₃H₄₄O₃) which is named Substance A, was purified from olive leaves infected withCycloconium oleaginum Cast. The mutagenic effect of this substance was detected using TA 100 and TA 102 strains ofSalmonella in the Ames test usingBacillus subtilis strains M45 rec^–, H17 rec⁺ in the rec assay. Another substance manifesting the mutagenic effect was found in the extract from theCycloconium oleaginum culture. This substance was not detected in the extract from contaminated olive leaves. Substance A increased electrolytes leakage from tissue of olive leaves, thus manifesting its phytotoxicity.     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

Renaturation of a reduced Taiwan cobra cardiotoxin.

Refolding of a denatured protein obtained by reducing cardiotoxin from the Taiwan cobra with mercaptoethanol has been carried out in aqueous and non-aqueous solutions. Oxidation of the reduced protein in 0.05 M phosphate buffer (pH 7.2) resulted in isolating an active protein which showed, as compared to native cardiotoxin, identical conformation and biological activities such as lethality, antigenicity and muscle contracture inducing activity. On the other hand, the reduced protein was undergoing incorrect SS-pairing and several inactive products were formed in a mixture of 1,2-ethanediol and 1-propanol (1 : 1; v/v).     

Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins.

ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala). In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment.     

A Revision of the Genus Leptomischus Drake

In this revision, three species are recognised. A discussion is made on merging the genus Polysolenia into Leptomischus. Indopolysolenia burmanica Deb et Rout is reduced to a synonum of Leptomischus primuloides Drake. A key to species is given below: l. Flowers larger, with corollas more than 2cm long. 2.Stipules 3-fid or torn; Leaves narrowly lanceolate; stigmas with 2 short obtuse lobes ...................................................... 1.L. wallichii 2.Stipules entire; leaves obovate to elliptic; stigmas with 2 slender elongate lobes ......................................................... 2. L. primuloides 1.Flowers smaller, with corollas 6-6.5mm long; stigmas with 2 lanceolate lobes .......................................................... 3. L. parviflorus