Reflections on the Conservation and Sustainable Use of Genetic Resources in the Deep Seabed Beyond the Limits of National Jurisdiction

Attention is increasingly being given to genetic resources in the deep seabed beyond the limits of national jurisdiction owing to their considerable potential scientific and economic value. At the same time, there are concerns that the increased demand for these genetic resources may result in their unsustainable collection or even in the extinction of species in the deep seabed. At present there is no specific legal framework governing these resources in international law. Thus, this article explores the relevant rules of international law applicable to the conservation and sustainable use of genetic resources in the deep seabed.     

Cloning of a Vero toxin (VT2) gene from a VT2-converting phage isolated from Escherichia coli 0157: H7

Abstract A Vero toxin (VT2 or Shiga-like toxin II)-converting phage was isolated from Escherichia coli 0157: H7 strain J-2. Nontoxigenic E. coli C600 produced VT2 when lysogenized with the toxin-converting phage. Eco RI fragments of the phage DNA were ligated with Eco RI-digested pBR322 or pUC118 and were transformed into E. coli MC1061 or MV1184. Transformants exhibiting VT2 production commonly contained a 4.6 kb Eco RI fragment. It was found that a 2.3 kb Kpn I- Sph I fragment coded VT2 production and that this fragment hybridized weakly with the 2.1 kb fragment encoding VT1.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

Stain Technology: A Simplified Aluminum Stain in Paraffin Sections of Bone from Hemodialysis Patients

A new simplified method has been devised for staining aluminum and has been tested in paraffin sections of bone from 60 patients who have undergone hemodialysis. Iliac crest bone fragments were fixed in 20% phosphate-buffered formalin for less than a day and demineralized at room temperature in 10% phosphate-buffered formalin containing 5% formic acid for only 2 to 3 hr. Four-micron paraffin sections, accompanied by positive controls, were stained with Maloney's aluminum stain, the Berlin blue reaction for iron, dylon or Congo red for amyloid and von Kossa's reaction for calcium. Aluminum and iron were demonstrated particularly at the mineralizing front of bony tissues; aluminum in 52 cases, iron in 45. Dylon staining also gave positive results in 52 cases. It is important in determining whether aluminum deposition is present that the von Kossa reaction remains positive even after demineralization. This method may be more useful for demonstrating aluminum in bony tissues than the complicated and time-consuming resin-embedding method currently used.     

ATP-Dependent Depolymerization of Cortical Microtubules by an Extract in Tobacco BY-2 Cells

An extract of tobacco BY-2 cells, prepared by rupturing protoplastsby exposure to centrifugal force, depolymerized cortical microtubuleson protoplast ghosts of BY-2 cells in an ATP-dependent manner.Depolymerization of cortical microtubules by the cell extractdid not occur in the presence of taxol. The extract failed todepolymerize microtubules of phragmoplasts and spindles isolatedfrom BY-2 cells. The activity was detected in a fraction precipitatedbetween 30–50% saturation with ammonium sulfate. Theseresults suggest the presence in the cell extract of a proteinfactor(s) that depolymerizes cortical microtubules in an ATP-dependentmanner. The importance of this factor(s) in the premitotic disappearanceof cortical microtubules is discussed. (Received April 25, 1990; Accepted September 6, 1990)     

Analysis of Vibrio cholerae O139 Bengal Isolated from Different Geographical Areas Using Macrorestriction DNA Analysis

Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains. NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns. Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity. These results further reiterate the spread of an identical clone of V. cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.