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Deep ocean environmental biotechnology

Major recent advances in deep-sea biotechnology have come in the form of continuing discoveries of novel microorganisms, unexpected genetic diversity, and new natural products of potential relevance to human health or environmental bioremediation. Continuing explorations of submarine hydrothermal vent environments have yielded new hyperthermophiles (maximal growth at 90 degreesC or greater) and more evidence that elevated hydrostatic pressure stabilizes cells and enzymes at high temperature. Vent samples have also yielded new mesophiles (optimal growth near 30 degreesC) that produce heparin-like exopolysaccharides or express extraordinary tolerance (removal by precipitation) of heavy metals. From the cold deep sea have come new findings of unexpected microbial diversity and the promise of industrially useful enzymes or secondary metabolites. New classes of predictive models are emerging to guide future exploration of microbial diversity in the deep ocean.

Journal Article↗

Dynamics of shear-transformation zones in amorphous plasticity: energetic constraints in a minimal theory.

We use energetic considerations to deduce the form of a previously uncertain coupling term in the shear-transformation-zone (STZ) theory of plastic deformation in amorphous solids. As in the earlier versions of the STZ theory, the onset of steady deformation at a yield stress appears here as an exchange of dynamic stability between jammed and plastically deforming states. We show how an especially simple "quasilinear" version of this theory accounts qualitatively for many features of plasticity such as yielding, strain softening, and strain recovery. We also show that this minimal version of the theory fails to describe certain other phenomena, and argue that these limitations indicate needs for additional internal degrees of freedom beyond those included here.

Journal Article↗

Biomechanical comparison of orthofix pins and cortical bone screws in a canine humeral condylar fracture model.

OBJECTIVE: To compare shear stability of simulated humeral lateral condylar fractures reduced with either a self-compressing pin or cortical bone screw. STUDY DESIGN: In vitro biomechanical tests. SAMPLE POPULATION: Bilateral cadaveric canine humeri (n=18) without evidence of elbow disease. METHODS: Lateral condylar fracture was simulated by standardized osteotomy. Bone fragments were stabilized with a self-compressing pin or a cortical bone screw (2.7 or 3.5 mm) inserted in lag fashion. Specimens were mounted in a materials testing system and the condylar fragment displaced in a proximal direction until failure. Mechanical testing variables derived from load-deformation curves were compared between stabilization methods using a Student's paired t-test. RESULTS: There were no statistically significant differences for mechanical testing variables between pin and screw stabilized specimens at expected walk and trot loads. Three yield points subjectively coincided with yield of the interfragmentary interface (Y1), bone at the implant interface (Y2), and implant deformation (Y3). Displacements at Y1 were 48-156% greater for pin than screw stabilized specimens. Y2 and Y3 loads were higher for screw than pin stabilized specimens, but likely supraphysiologic for dogs convalescing after surgical repair. CONCLUSIONS: A self-compressing pin or a cortical bone screw inserted in lag fashion both provided adequate strength in applied shear to sustain expected physiologic loads through the repaired canine elbow during postoperative convalescence. CLINICAL RELEVANCE: Because self-compressing pins were easy to implant and mechanical properties were not significantly different than cortical screws at expected physiologic loads, pins should be considered for the repair of traumatic humeral condylar fractures.

Animals↗

Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum.

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.

Animals↗

Inactivation and stabilization of IgA protease from the human oral bacterium Streptococcus sanguis.

The oral bacterium, Streptococcus sanguis, secretes an extracellular protease which specifically cleaves immunoglobulin A (IgA). During in vitro growth of Strep. sanguis at 37 degrees C the IgA protease was completely inactivated within 2 h. Lowering the growth temperature to 31 degrees C reduced the inactivation rate 50 per cent and a doubling of enzyme yield was obtained. The IgA protease could be stabilized by the presence of whole human saliva, by a low concentration of IgA, or by inhibition of non-specific protease activity. Therefore, the inactivation of IgA protease appeared to be the cumulative result of thermal denaturation and inactivation by non-specific proteases.

Humans↗

Procurement of radioiodinated glucose derivative and its biological character.

Radiolabeled glucose derivatives are attracting great interest in the clinical field. Development of an analogous substrate labeled with a practical radionuclide, 123I, is most desirable, however, no radioiodine labeled glucose derivative has been reported as being chemically and biologically compatible. Thus, in the present study, a glucose derivative substituted at the C-2 position by a p-iodobenzyl group, a 2-O-(p-iodobenzyl)-D-glucose (IBG) was designed and its synthesis was carried out. A very easy and simple synthetic method was developed, and the obtained IBG showed appropriate purity and stability for its radioiodination. [125I]IBG was obtained by radioisotopic exchange reaction with high radiochemical purity and radiochemical yield, requiring no purification. The good in vivo and in vitro stability and the chemical and biological characteristic displayed by the new [125I]IBG stimulated the measurement of the brain uptake index (BUI). In the presence of glucose, brain uptake inhibition was detected, a good indication of a glucose carrier mediator for the transport of [125I]IBG through the blood-brain barrier, a similar feature to that of [14C]glucose or 3-O-[14C]methylglucose. The newly designed ligand IBG holds good promise for the study of regional cerebral glucose utilization, should the 123I become available.

Animals↗

[Adaptation of transgenic strains of insect-resistance cotton to different ecological environments].

Seven transgenic strains of insect-resistance cotton, WH1, WH2, WH3, WH4, WH5, WH6 and BG2, and one common strain Simian-3 (CK) were selected to investigate their adaptation to insect-resistance and yield trait at 10 different sites. The results show that all transgenic strains had a strong resistance to bollworm(Helicoverpa armigera) and pink bollworm (Pectinophora gossypiella), with a high stability and no difference in 10 sites, but the yield trait was significantly influenced by environmental factors. The interaction between strains and sites was significant. In comparison with CK, WH6 and BG2 had a good insect-resistance and high fiber yield, and could be applied in practice.

Adaptation, Physiological↗

D-aspartate oxidase from beef kidney. Purification and properties.

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.

Amino Acid Oxidoreductases↗

Layered double hydroxide stability. 1. Relative stabilities of layered double hydroxides and their simple counterparts.

Solutions containing di- and trivalent metal chlorides [M(II) = Mg2+, Zn2+, Co2+, Ni2+, Mn2+; M(III) = Al3+, Fe3+] were titrated with NaOH to yield hydrotalcite-like layered double hydroxides (LDH), [[M(II)]1-x[M(III)]x(OH)2][Cl]x yH2O, by way of M(III) hydroxide/hydrous oxide intermediates. Analysis of the resultant titration curves yields nominal solubility constants for the LDH. The corresponding LDH stabilities are in the order Mg < Mn < Co approximately Ni < Zn for M(II) and Al < Fe for M(III). The stability of LDH relative to the separate metal hydroxides/hydrous oxides is discussed.

Aluminum↗

Role of hydrophobic effects in the reaction of a polynuclear aromatic diol epoxide with oligodeoxynucleotides in aqueous solutions.

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[alpha]pyrenediol epoxide-oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r, 8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[alpha] pyrene (BPDE) and the single-stranded oligonucleotide 5'-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above approximately 3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10-40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE-dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[alpha]pyrene as a model system.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

Nuclear isomers in superheavy elements as stepping stones towards the island of stability.

A long-standing prediction of nuclear models is the emergence of a region of long-lived, or even stable, superheavy elements beyond the actinides. These nuclei owe their enhanced stability to closed shells in the structure of both protons and neutrons. However, theoretical approaches to date do not yield consistent predictions of the precise limits of the 'island of stability'; experimental studies are therefore crucial. The bulk of experimental effort so far has been focused on the direct creation of superheavy elements in heavy ion fusion reactions, leading to the production of elements up to proton number Z = 118 (refs 4, 5). Recently, it has become possible to make detailed spectroscopic studies of nuclei beyond fermium (Z = 100), with the aim of understanding the underlying single-particle structure of superheavy elements. Here we report such a study of the nobelium isotope 254No, with 102 protons and 152 neutrons--the heaviest nucleus studied in this manner to date. We find three excited structures, two of which are isomeric (metastable). One of these structures is firmly assigned to a two-proton excitation. These states are highly significant as their location is sensitive to single-particle levels above the gap in shell energies predicted at Z = 114, and thus provide a microscopic benchmark for nuclear models of the superheavy elements.

Journal Article↗

Interaction of the anticancer agent Taxol (paclitaxel) with phospholipid bilayers.

Taxol (paclitaxel), a promising agent for use in ovarian and breast cancer, was incorporated into lipid vesicles (liposomes) composed of different saturated and unsaturated phosphatidylcholines, as well as saturated phosphatidylcholines mixed with the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) at different molar ratios, to yield information about Taxol-liposome interactions. For the physicochemical characterization of the thermodynamic, structural, and dynamic properties of these mixtures, differential scanning calorimetry (DSC), steady-state fluorescence depolarization, and Fourier transform IR spectroscopy was used. Time-dependent DSC measurements on 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/Taxol mixtures of different concentrations were performed to yield information on the long-term stability of Taxol-liposome complexes. Partitioning of Taxol into saturated lipid bilayers results in changes of membrane physical properties, such as phase transition temperatures and lipid order parameter, that are different from those observed for unsaturated and charged phospholipid bilayers. Taxol incorporated into saturated phospholipids changes their thermotropic phase behavior: it reduces the lipid order parameter (i.e., has a "fluidizing" effect) in the gel phase of the lipid bilayers. On the contrary, partitioning of Taxol into unsaturated fluid phospholipid bilayers has a slight "rigidization" effect. The saturated lipid bilayer systems DPPC and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/DMPS have been identified with the highest incorporation efficiency for Taxol and are thus candidates for drug vehicles that can improve the therapeutic efficacy of Taxol.

Antineoplastic Agents, Phytogenic↗

DNA-polycation complexation and polyplex stability in the presence of competing polyanions.

Polyelectrolyte complex (polyplex) formation was studied by employing tapping mode atomic force microscopy (AFM) and an ethidium bromide fluorescence assay. The polycations chitosan and poly-L-lysine were used to compact DNA and the stability of the polyplexes was evaluated upon exposure to competing polyanions (alginate and xanthan). Furthermore, the relative preference of these polycations for DNA and the competing polyanion was investigated. The results showed that neither poly-L-lysine nor chitosan displayed any selectivity in binding to DNA relative to the competing polyanions, demonstrating the importance of electrostatics in the binding of a polycation to a polyanion. However, the ability of the polyanions to destabilize the DNA-polycation complexes depended on both the polyanion and the polycation employed, indicating that polymer-specific properties are also important for the complexation behavior and polyplex stability. Destabilization experiments further showed that annealing yielded complexes that were less prone to disruption upon subsequent exposure to alginate. Annealing experiments of plasmid DNA-chitosan complexes showed an increased fraction of rods following temperature treatment, indicating that the rods most likely are the more stable morphology for this system.

Alginates↗

Molecular interactions between the photoreceptor G protein and rhodopsin.

1. The visual transduction system of the vertebrate retina is a well-studied model for biochemical and molecular studies of signal transduction. The structure and function of rhodopsin, a prototypical G protein-coupled receptor, and transducin or Gt, the photoreceptor G protein, have been particularly well studied. Mechanisms of rhodopsin-Gt interaction are discussed in this review. 2. The visual pigment rhodopsin contains a chromophore, and thus conformational changes leading to activation can be monitored spectroscopically. A model of the conformational changes in the activated receptor is presented based on biophysical and biochemical data. 3. The current information on sites of interaction on receptors and cognate G proteins is summarized. Studies using synthetic peptides from amino acid sequences corresponding to Gt and rhodopsin have provided information on the sites of rhodopsin-Gt interaction. Synthetic peptides from the carboxyl terminal region of alpha t mimic Gt by stabilizing the active conformation of rhodopsin, Metarhodopsin II. 4. The conformation of one such peptide when it is bound to Metarhodopsin II was determined by 2D NMR. The model based on the NMR data was tested using peptide analogs predicted to stabilize or break the structure. These studies yield molecular insight into why toxin-treated and mutant G proteins are uncoupled from receptors.

Amino Acid Sequence↗

Resolution of human fibroblast interferon into two distinct classes by thiol exchange chromatography.

Chromatography of crude and purified human fibroblast interferon on activated thiol Sepharose 4B yielded two classes of material exhibiting different stability to inactivation by mechanical stress. Interferon which bound to the gel accounted for 20--30 per cent of recoverable activity, could be eluted with reducing agents and was unstable. Unbound material did not bind on subsequent rechromatography and was stable to shear forces. Leucocyte interferon did not bind, nor did fibroblast interferon treated with thiol blocking reagents. Peak eluted fractions had a specific activity of up to 10(7.9) units per milligram of protein, representing a 1700 fold purification.

Chloromercuribenzoates↗

EPR properties of mixed-valent mu-oxo and mu-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K.

Radiolytic reduction at 77 K of oxo/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S = 1/2 ground state with small g-anisotropy as characterized by the uniaxial component (gz-gav/2 < 0.03) observable at temperatures as high as approximately 100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (gz-gav/2 > 0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins.

Anisotropy↗

Agronomic performance and transcriptional analysis of carotenoid biosynthesis in fruits of transgenic HighCaro and control tomato lines under field conditions.

Genetic manipulation of carotenoid biosynthesis in higher plants has been the objective of a number of biotechnology programs, e.g. the Golden Rice Program. However, tomato (Solanum lycopersicum L.), which naturally accumulates lycopene in fruits, has attracted the attention of many groups who have manipulated it to increase or diversify carotenoid accumulation. One of the most significant achievements was "HighCaro (HC)," a transgenic tomato plant constitutively expressing the tomato lycopene beta-cyclase (tLcy-b), that produces orange fruits due to the complete conversion of lycopene to beta-carotene. In this article we report the results of a field trial conducted in Metaponto (Italy) on HC and on two control genotypes to evaluate the stability of the transgenic trait and their yield performances. Transcriptional regulation of eight genes involved in carotenogenesis was assayed by quantitative real-time PCR (qRT-PCR) analysis on fruits collected at four distinct development stages. Statistical analysis results demonstrated that in field conditions the transgene maintained its ability to induce the conversion of lycopene to beta-carotene. Moreover, agronomic performances and fruit quality in the transgenic line were not impaired by this metabolic disturbance. Results of qRT-PCR analysis suggested that transcription of PSY-1, PDS and ZDS genes were developmentally regulated in both genotypes. Unexpectedly, Lcy-b expression in transgenic fruits was also developmentally regulated, despite the fact that the gene was driven by a constitutive promoter. Our data provide evidence that in photosynthetic cells a strict and aspecific mechanism controls the level of transcripts until the onset of chromoplasts differentiation, at which point a gene-specific control on transcription takes place.

Agriculture↗

Immobilization of L-glutamate dehydrogenase into soluble cross-linked polymers. ADP effect and electron microscopy studies.

Active soluble cross-linked L-glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) albumin polymers were produced. Electron microscopic studies and kinetic properties were studied with the polymer in solution and compared with previous published data about the enzyme immobilized inside proteic films (Barbotin, J.N. and Breuil, M. (1978) Biochim. Biophys. Acta 525, 18--27). The glutaraldehyde effect on activity yield, ADP and beta-NAD+ protection, stability and pH rate profile were studied and discussed. Apparent Michaelis constants were determined with soluble polymers produced with or without ADP during the grafting process. Experiments were performed on the regulatory properties of immobilized glutamate dehydrogeanse showing the decrease of ADP activation and GTP inhibition as compared to the free form. In other respects, electron microscopy observations showed morphological differences between the two populations of soluble polymers produced in presence of ADP, obtained after gel filtration on Sepharose 6B. Linear aggregates of high molecular weight and classical soluble polymers were obtained. Similar Km values and regulatory properties were exhibited by the two forms, demonstrating the absence of interdependence between the allosteric control and the polymerization of enzyme monomers.

Adenosine Diphosphate↗