Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 → F Mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to Generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K-m toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K-m was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide: acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K-d for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

Vitamin B12 stalls the 80 S ribosomal complex on the hepatitis C internal ribosome entry site

The effect of cyanocobalamin (CNCbl, vitamin 1312) on hepatitis C virus internal ribosome entry site (HCV IRES)-dependent initiation of translation was studied by ribosomal toeprinting and sucrose gradient centrifugation analysis. These results suggested that CNCbl did not inhibit HCV IRES-dependent translation by a competitive binding mechanism. CNCbl allowed 80 S elongation complex formation on the mRNA, but stalled the initiation at that point, effectively trapping the 80 S ribosomal complexes on the HCV TRES. CNCbl had no effect on cap-dependent mRNA, consistent with the known mRNA specificity of this translational inhibitor. To help elucidate the mechanism, comparative data were collected for the well-characterised translation inhibitors cycloheximide and 5'-guanylyl-imidophosphate, Although CNCbl stalled HCV IRES-dependent translation at approximately the same step in initiation as cycloheximide, the mechanisms of these two inhibitors are distinct. (C) 2002 Elsevier Science Ltd. All rights reserved.

Analysis of the sequence and expression of a second putative acetylcholinesterase cDNA from organophosphate-susceptible and organophosphate-resistant cattle ticks

The cattle tick, Boophilus microplus, is a major pest of cattle in Australia, Central and South America, and parts of Africa and Asia. Control of ticks with organophosphates (OPs) and carbamates, which target acetylcholinesterases (AChE), led to evolution of resistance to these pesticides. Alleles at the locus studied here, AChE2, from OP-susceptible female ticks from Australia and Mexico differed at 46 of 1689 nucleotide positions (20 putative amino acid differences) whereas alleles from three strains of OP-resistant ticks from Australia differed with the allele from the Australian susceptible ticks at six to 13 nucleotide positions (three to six putative amino acid differences). However, the role, if any, of these polymorphisms in the OP-resistance phenotype is unknown. Certainly none of the polymorphisms correspond to sites in ACK that are involved in catalysis or binding of acetylcholine in other organisms. Both of the AChE loci of B. microplus, AChE1 and AChE2, are apparently expressed in synganglia; AChE1 is also expressed in salivary glands and ovaries, in OP-susceptible and OP-resistant ticks. This seems to contradict studies of enzyme kinetics, which indicated that only one form of AChE was present in the synganglia, the site of the action of OPs, in this species of tick. (C) 2002 Elsevier Science Ltd. All rights reserved.

U-Series dating of Liujiang hominid site in Guangxi, Southern China

It has been established that modern humans were living in the Levant and Africa ca. 100 ka ago. Hitherto, this has contrasted with the situation in China where no unequivocal specimens of this species have been securely dated to more than 30 ka. Here we present the results of stratigraphic studies and U-series dating of the Tongtianyan Cave, the discovery site of the Liujiang hominid, which represents one of the few well-preserved fossils of modern Homo sapiens in China. The human fossils are inferred to come from either a refilling breccia or a primarily deposited gravel-bearing sandy clay layer. In the former case, which is better supported, the fossils would date to at least similar to 68 ka, but more likely to similar to 111-139 ka. Alternatively, they would be older than, similar to 153 ka. Both scenarios would make the Liujiang hominid one of the earliest modem humans in East Asia, possibly contemporaneous with the earliest known representatives from the Levant and Africa. Parallel studies on other Chinese localities have provided supporting evidence for the redating of Liujiang, which may have important implications for, the origin of modem humans. (C) 2002 Elsevier Science Ltd. All rights reserved.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

Elucidating the molecular mechanisms that underlie the target control of motoneuron death

Approximately half of the motoneurons generated during normal embryonic development undergo programmed cell death. Most of this death occurs during the time when synaptic connections are being formed between motoneurons and their target, skeletal muscle. Subsequent muscle activity stemming from this connection helps determine the final number of surviving motoneurons. These observations have given rise to the idea that motoneuron survival is dependent upon access to muscle derived trophic factors, presumably through intact neuromuscular synapses. However, it is not yet understood how the muscle regulates the supply of such trophic factors, or if there are additional mechanisms operating to control the fate of the innervating motoneuron. Recent observations have highlighted target independent mechanisms that also operate to support the survival of motoneurons, such as early trophic-independent periods of motoneuron death, trophic factors derived from Schwann cells and selection of motoneurons during pathfinding. Here we review recent investigations into motoneuron cell death when the molecular signalling between motoneurons and muscle has been genetically disrupted. From these studies, we suggest that in addition to trophic factors from muscle and/or Schwann cells, specific adhesive interactions between motoneurons and muscle are needed to regulate motoneuron survival. Such interactions, along with intact synaptic basal lamina, may help to regulate the supply and presentation of trophic factors to motoneurons.

Using siting algorithms in the design of marine reserve networks

Using benthic habitat data from the Florida Keys (USA), we demonstrate how siting algorithms can help identify potential networks of marine reserves that comprehensively represent target habitat types. We applied a flexible optimization tool-simulated annealing-to represent a fixed proportion of different marine habitat types within a geographic area. We investigated the relative influence of spatial information, planning-unit size, detail of habitat classification, and magnitude of the overall conservation goal on the resulting network scenarios. With this method, we were able to identify many adequate reserve systems that met the conservation goals, e.g., representing at least 20% of each conservation target (i.e., habitat type) while fulfilling the overall aim of minimizing the system area and perimeter. One of the most useful types of information provided by this siting algorithm comes from an irreplaceability analysis, which is a count of the number of, times unique planning units were included in reserve system scenarios. This analysis indicated that many different combinations of sites produced networks that met the conservation goals. While individual 1-km(2) areas were fairly interchangeable, the irreplaceability analysis highlighted larger areas within the planning region that were chosen consistently to meet the goals incorporated into the algorithm. Additionally, we found that reserve systems designed with a high degree of spatial clustering tended to have considerably less perimeter and larger overall areas in reserve-a configuration that may be preferable particularly for sociopolitical reasons. This exercise illustrates the value of using the simulated annealing algorithm to help site marine reserves: the approach makes efficient use of;available resources, can be used interactively by conservation decision makers, and offers biologically suitable alternative networks from which an effective system of marine reserves can be crafted.

Stock discrimination and movements of narrow-barred Spanish mackerel across northern Australia as indicated by parasites

The parasite fauna of Spanish mackerel Scomberomorus commerson from 10 sites across northern Australia and one site in Indonesia, was examined to evaluate the degree of movement and subsequent stock structure of the fish. Kupang fish (Indonesia) had very few Terranova spp.. Grillotia branchi, Otobothrium cysticum or Pterobothrium sp. compared to Australian fish, indicating that no Australian fish enter the Kupang fishery. Univariate and discriminant function analysis of four 'temporary' parasite species, the copepod Pseudocyenoides armatus and the monogeneans Gotocotyla bivaginalis, Pricea multae and Pseudothoracocotyla ovalis, demonstrated little similarity between areas of northern Australia, indicating minimal short-term exchange between neighbouring groups of S. commerson. Analyses of five 'permanent' parasite species, the larval helminths G. branchi, O. cysticum, Pterobothrium sp., Callitetrarhynchus gracilis and Paranybelinia balli, also revealed large differences between areas thus indicating long-term separation. There are at least six parasitological stocks across northern Australia: Fog Bay/Bathurst Island, Cape Wessel. Groote/Sir Edward Pellew. Mornington Island, Weipa. and the Torres Strait. The occurrence of a few irregular fish in the samples suggested that LIP to 5% of fish moved between stocks during their lifetime. The similarity of within-school variability to that between schools showed that the fish do not form long-term school associations. (C) 2003 The Fisheries Society of the British Isles.

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branchedchain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Angstrom resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K-i of 3.3 nM blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.

Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.

Site-directed mutagenesis of the ATM promoter: Consequences for response to proliferation and ionizing radiation

Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. (C) 2003 Wiley-Liss, Inc.

Winding patterns for actively shielded shim coils with asymmetric target-fields

A method is presented for calculating the currents and winding patterns required to design independent zonal and tesseral shim coils for magnetic resonance imaging. Both actively shielded and unshielded configurations are considered, and the region of interest can be located asymmetrically with respect to the coil's length. Streamline, target-field and Fourier-series methods are utilized. The desired target-field is specified at two cylindrical radii, on and inside a circular conducting cylinder of length 2L and radius a. The specification is over some asymmetric portion pL < z < qL of the coil's length (-1 < p < q < 1). Arbitrary functions are used in the outer sections, -L < z < pL and qL < z < L, to ensure continuity of the magnetic field across the entire length of the coil. The entire field is then periodically extended as a half-range cosine Fourier series about either end of the coil. The resultant Fourier coefficients are then substituted into the Fourier-series expressions for the internal and external magnetic fields, and current densities and stream functions on both the primary coil and shield. A contour plot of the stream function directly gives the required coil winding patterns. Spherical harmonic analysis and shielding analysis on field calculations from a ZX shim coil indicate that example designs and theory are well matched.

A novel target-field method for magnetic resonance shim coils: III. Shielded zonal and tesseral coils

This paper continues the development of a new approach for the design of shim and gradient coils, used in magnetic resonance imaging (MRI) applications. A cylindrical primary coil of radius a and length 2L is placed inside a co-axial shield cylinder of radius b. An active shielding strategy is used to create a desired target field at an arbitrarily specified (cylindrical) location within the primary coil, and to annul the field at a certain radius outside the shield. The form of the interior target field may be chosen arbitrarily by the designer, although zonal and tesseral harmonics are typically used in MRI applications. The method presented here designs coil windings on both the primary and shielding cylinders, to produce fields that conform to the specified interior target field and the annulled field exterior to the shield. An additional feature of the method presented here is that the target field inside the primary coil is matched at two different radii, to improve overall accuracy. The method is illustrated by designing several shielded shim coils, for creating higher order tesseral fields located asymmetrically within the coil. The simpler case of pure zonal fields is discussed separately and applied to the design of some higher order shielded coils.