Use of lablab (Lablab purpureus (L.) Sweet) for bio-control by native arthropods and its effect on yield of pumpkins.

Silverleaf whitefly (SLW, Bemisia tabaci MEAM1) and aphids are sap-sucking insects, which pose a serious threat to Australian cucurbit crops and the horticulture industry. Traditional chemical control for these insect pests is becoming less effective, and there is a need to search for alternative or supplementary methods. This study aimed to manipulate the habitat of pumpkin crops in a tropical setting (Queensland, Australia), by growing pumpkins (var. Japanese pumpkin) alone and between lablab (Lablab purpureus L. Sweet). It was hypothesized that the presence of lablab will increase the populations of natural enemies, and through their control of insect pests such as SLW and aphids, will affect pumpkin yield. The population of arthropods (natural enemies and pests of pumpkin), with a focus on SLW and aphids, were sampled weekly on both lablab and pumpkin crop for a total of 21 weeks. Results showed that lablab hosted more enemies of SLW per plant than pumpkin in either treatment. In addition, adult SLW numbers were significantly higher in the pumpkin-only crop compared with the pumpkin grown between lablab, while pumpkin in the mixed plantings had significantly more ladybirds and lacewing larvae (P < 0.05). While there was no significant difference in the average fruit weight between treatments, the total weight (kg) and number of marketable pumpkins per hectare was greater (P < 0.05) for the pumpkin/lablab treatment than the pumpkin-only treatment. This study shows that growing lablab alongside a pumpkin crop may enhance natural enemies of SLW and could significantly increase the yield.

Vessel generator noise as a settlement cue for marine biofouling species.

Underwater noise is increasing globally, largely due to increased vessel numbers and international ocean trade. Vessels are also a major vector for translocation of non-indigenous marine species which can have serious implications for biosecurity. The possibility that underwater noise from fishing vessels may promote settlement of biofouling on hulls was investigated for the ascidian Ciona intestinalis. Spatial differences in biofouling appear to be correlated with spatial differences in the intensity and frequency of the noise emitted by the vessel's generator. This correlation was confirmed in laboratory experiments where C. intestinalis larvae showed significantly faster settlement and metamorphosis when exposed to the underwater noise produced by the vessel generator. Larval survival rates were also significantly higher in treatments exposed to vessel generator noise. Enhanced settlement attributable to vessel generator noise may indicate that vessels not only provide a suitable fouling substratum, but vessels running generators may be attracting larvae and enhancing their survival and growth.

Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise.

Underwater sound plays an important role in the settlement behaviour of many coastal organisms. Large steel-hulled vessels are known to be a major source of underwater sound in the marine environment. The possibility that underwater sound from vessels may promote biofouling of hulls through triggering natural larval settlement cues was investigated for the mussel, Perna canaliculus. The mussel larvae showed significantly faster settlement when exposed to the underwater noise produced by a 125-m long steel-hulled passenger and freight ferry. Median time to attachment on the substrata (ie settlement) was reduced by 22% and the time taken for all experimental larvae to settle was reduced by 40% relative to a silent control. There was no difference in the survival of the mussel larvae among the various noise treatments. The decrease in settlement time of the mussel larvae appeared to correlate with the intensity of the vessel sound, suggesting that underwater sound emanating from vessels may be an important factor in exacerbating hull fouling by mussels.

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments.

AIMS: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. METHODS AND RESULTS: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. CONCLUSIONS: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l(-1)), streptomycin/penicillin (25 mg l(-1)) and nystatin (10 mg l(-1)) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.

Natural J-coupling analysis: interpretation of scalar J-couplings in terms of natural bond orbitals.

The natural J-coupling (NJC) method presented here analyzes the Fermi contact portion of J-coupling in the framework of finite perturbation theory applied to ab initio/density function theory (DFT) wave functions, to compute individual and pairwise orbital contributions to the net J-coupling. The approach is based on the concepts and formalisms of natural bond orbital (NBO) methods. Computed coupling contributions can be classified as Lewis (individual orbital contributions corresponding to the natural Lewis structure of the molecule), delocalization (resulting from pairwise donor-acceptor interactions), and residual repolarization (corresponding to correlation-like interactions). This approach is illustrated by an analysis of the angular and distance dependences of the contributions to vicinal (3)J(HH) couplings in ethane and to the long-range (6)J(HH) couplings in pentane. The results indicate that approximately 70% or more of the net J-coupling is propagated by steric exchange antisymmetry interactions between Lewis orbitals (predominantly sigma bonding orbitals). Hyperconjugative sigma to sigma delocalization interactions account for the remainder of the coupling. Calculated pairwise-steric and hyperconjugative-delocalization energies provide a means for relating coupling mechanisms to molecular energetics. In this way, J-coupling contributions can be related directly to the localized features of the molecular electronic structure in order to explain measured J-coupling patterns and to predict J-coupling trends that have yet to be measured.

Three-dimensional structure of the vacuolar ATPase proton channel by electron microscopy.

Vacuolar ATPases are ATP hydrolysis-driven proton pumps found in the endomembrane system of eucaryotic cells where they are involved in pH regulation. We have determined the three-dimensional structure of the proton channel domain of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy at 21 A resolution. The model shows an asymmetric protein ring with two small openings on the luminal side and one large opening on the cytoplasmic side. The central hole on the luminal side is covered by a globular protein, while the cytoplasmic opening is covered by two elongated proteins arranged in a collar-like fashion.

Structure of the vacuolar adenosine triphosphatases.

Vacuolar adenosine triphosphatases (V-ATPases) represent an important class of proton pumps found in endomembrane systems of eucaryotic cells, where they are involved in pH regulation. Progress has been made in the structure determination of this large, membrane-bound multisubunit enzyme complex. Electron microscopy of the V-ATPase has revealed a ball-and-stalk-like structure similar to F1F0-type ATP synthase, to which the V-ATPase is evolutionary related. Aside from the overall structural similarity of the V-ATPase and F-ATP synthase, a number of distinct structural differences exist between the two related enzymes, giving clues to their different function and regulation in the organism.

Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATPsynthase by immuno electron microscopy: the delta subunit binds on top of the F(1).

The binding site of the delta subunit in the F(1)F(0)-ATPsynthase from Escherichia coli has been determined by electron microscopy of negatively stained, antibody-decorated enzyme molecules. The images show that the antibody is bound at the very top of the F(1) domain indicating that at least part of delta is bound in the dimple formed by the N termini of the alpha and beta subunits. The data may explain why there is only one binding site for delta on the F(1) despite there being three identical alphabeta pairs. The finding also implies that the b subunits of the F(0) have to extend all the way from the membrane surface to the very top of the F(1) domain to make contact with the delta subunit.

F1F0-ATP synthase-stalking mind and imagination.

Electron microscopy together with image analysis has been used to study the structure of the intact F1F0-ATPsynthase from Escherichia coli. A procedure has been developed which allows preparation of detergent-free enzyme. Aside from the well known two-domain structure, images of F1F0 prepared by this procedure show a number of additional features, including a second stalk, which can be seen extending all the way from the F0 to the top of the F1 in some images, and a small protein on the very top of the F1, which has been identified as the delta subunit by decoration with a monoclonal antibody. In light of these results, a refined model of the subunit arrangement of the complex is presented.

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.

Recall of a lead-contaminated vitamin and mineral supplement in a clinical trial.

PURPOSE: The Treatment of Lead-exposed Children (TLC) trial tested whether developmental outcome differed between children treated for lead poisoning with succimer or placebo. On 7 July 1997, TLC was informed that the vitamin and mineral supplements it gave to all children were contaminated with about 35 microg of lead per tablet. METHODS: TLC recalled the contaminated supplements and measured the children's exposure. RESULTS: The families of 96% of the children were contacted with 30 days. Among the 571 children to whom the contaminated supplements were dispensed, the mean increase in blood lead was 0.06+/-0.01 micromol/L (1.2+/-0.2 microg/dL); among 78 children to whom they were not, it was 0.09+/-0.03 micromol/L (1.8+/-0.7 microg/dL). There was no evidence of a dose-response relation between estimated supplement consumption and increase in blood lead concentration. CONCLUSIONS: The children's blood lead concentrations were not detectably affected by the contamination. Since the association of cognitive delay with lead exposure is best described for blood lead, we believe that the trial's inference about the effect of drug therapy on lead induced cognitive delay should be unaffected.

The second stalk: the delta-b subunit connection in ECF1F0.

The ATP synthase F1F0 is the smallest molecular motor yet studied. ATP hydrolysis drives the rotary motion of the primary stalk subunits gamma and epsilon relative to the alpha 3 beta 3 part of F1. Evidence is reviewed to show that the delta and b subunits provide a second stalk that can act as a stator to facilitate these rotational movements.

Solution structure of the epsilon subunit of the F1-ATPase from Escherichia coli and interactions of this subunit with beta subunits in the complex.

The solution structure of the epsilon subunit of the Escherichia coli F1-ATPase has been determined by NMR spectroscopy. This subunit has a two-domain structure with an N-terminal 10-stranded beta sandwich and a C-terminal antiparallel two alpha-helix hairpin, as described previously (Wilkens, S., Dahlquist, F. W., McIntosh, L. P., Donaldson, L. W., and Capaldi, R. A. (1995) Nat. Struct. Biol. 2, 961-967). New data show that the two domains interact in solution in an interface formed by beta strand 7 and the very C-terminal alpha-helix. This interface involves only hydrophobic interactions. The dynamics of the epsilon subunit in solution were examined. The two domains are relatively tightly associated with little or no flexibility relative to one another. The epsilon subunit can exist in two states in the ECF1F0 complex depending on whether ATP or ADP occupies catalytic sites. Proteolysis of the epsilon subunit in solution and when bound to the core F1 complex indicates that the conformation of the polypeptide in solution closely resembles the conformation of epsilon when bound to the F1 in the ADP state. Chemical and photo-cross-linking show that the epsilon subunit spans and interacts with two beta subunits in the ADP state. These interactions are disrupted on binding of ATP + Mg2+, as is the interaction between the N- and C-terminal domains of the epsilon subunit.

Electron microscopic evidence of two stalks linking the F1 and F0 parts of the Escherichia coli ATP synthase.

The structure of monodisperse ATP synthase from Escherichia coli (ECF1F0) has been examined by electron microscopy after negative staining of specimens. The F1 part is seen to be connected by two stalks. One is more centrally located and includes the gamma and epsilon subunits. The second stalk, observed here in ECF1F0, is arranged peripherally. It probably contains the delta and b subunits which, in addition to gamma and epsilon, are required for binding of the F1 and F0 parts of the complex. Other novel features of the F1F0 complex can be discerned. There is a cap at the top of the F1 part at which the second stalk may bind. This likely includes N-terminal stretches of the three copies of the alpha subunit and a part of the delta subunit. The F0 part is clearly asymmetric. The presence of two stalks in the complex has important functional implications. There is good evidence that the more central stalk of gamma and epsilon subunits is a mobile domain that rotates to link the three catalytic sites on beta subunits in turn, with the proton channel of the F0 part. The second stalk of delta and b subunits is then the stator which makes this rotation possible.

Organization of HIV-1 capsid proteins on a lipid monolayer.

In an in vitro system that mimics the assembly of immature human immunodeficiency virus (HIV) particles, ordered arrays of HIV-1 capsid (CA) proteins encoded by the viral gag gene have been obtained by incubation of histidine-tagged capsid proteins (His-HIVCA) beneath lipid monolayers containing the nickel-chelating lipid, 1,2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'-N-(5-amino-1- carboxypentyl)iminodiacetic acid)propyl ether. The membrane-bound His-HIVCA proteins formed small crystalline arrays of primitive (p1) unit cells with dimensions of a = 74.2 A, b = 126.2 A, gamma = 89.3 degrees. The image-analyzed two-dimensional projection of His-HIVCA assemblies shows a cage-like lattice, consisting of hexamer and trimer units, surrounding protein-free cage holes. The hexamer-coordinated cage holes of 26.3-A diameter are spaced at 74. 2-A intervals: these distances, and the hexamer-trimer arrangement, are consistent with previous, lower resolution studies on immature HIV-1 virus particles produced in vivo. Additionally, HIV-1 matrix protein trimer unit structures align to the His-HIVCA trimer units such that residues previously shown to interact with the HIV-1 gp120/gp41 envelope protein complex are oriented toward the hexamer cage holes. Our results form a bridge between results from conventional methods for the analysis of HIV particle structure.

Solution structure of the N-terminal domain of the delta subunit of the E. coli ATPsynthase.

NMR studies of the delta subunit of the Escherichia coli F1F0-ATPsynthase reveal that it consists of an N-terminal six alpha-helix bundle and a less well ordered C terminus. Both domains are part of one of two separate connections between F1 and F0.

Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.