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The Andromeda galaxy (M31) is our closest neighbouring spiral galaxy, making it an ideal target for studying the physics of the interstellar medium in a galaxy very similar to our own. Using new observations of M31 at 4.76GHz by the C-Band All-Sky Survey (C-BASS), and all available radio data at $1^\circ$ resolution, we produce the integrated spectrum and put new constraints on the synchrotron spectral index and anomalous microwave emission (AME) from M31. We use aperture photometry and spectral modelling to fit for the integrated spectrum of M31, and subtract a comprehensive model of nearby background radio sources. The AME in M31 is detected at $3\sigma$ significance with a peak near 30GHz and flux density $0.27\pm0.09$Jy. The synchrotron spectral index of M31 is flatter than our own Galaxy at $\alpha = -0.66 \pm 0.03$ with no strong evidence of spectral curvature. The emissivity of AME, averaged over the total emission from M31 is lower than typical AME sources in our Galaxy, implying that AME is not uniformly distributed throughout M31 and instead is likely confined to sub-regions -- this will need to be confirmed using future higher resolution observations around 20--30GHz., Comment: 16 pages, 6 figures. Accepted by MNRAS

Social science research has focused on hot spots of adolescent violence in marginalized urban neighborhoods for nearly a century. In contrast, in this study, we explore under-resourced urban areas that do not experience high rates of adolescent violence: "pockets of peace." We use a mixed-method approach to identify the sociodemographic, geographic, and criminological commonalities and differences between pockets of peace and other areas of concentrated disadvantage dealing with high rates of adolescent violence in Indianapolis, IN. More than one out of every ten of Indianapolis' areas of concentrated disadvantage meet the criteria to be labeled "pockets of peace." Quantitative data indicate that these areas have fewer prosocial institutions and experience lower homeownership rates than comparison under-resourced areas, and qualitative data point toward rental stability and residential longevity as potentially salient social factors within these contexts. As an alternative to using statistics to control for the context of structural disadvantage, studying pockets of peace and other "cold-spots" of adolescent violence presents an opportunity to understand community-level resilience within the real, lived context of structural disadvantage.

Objectives Ultrasound-guided regional anesthesia (UGRA) can be a powerful tool in the treatment of painful conditions commonly encountered in emergency medicine (EM) practice. UGRA can benefit patients while avoiding the risks of procedural sedation and opioid-based systemic analgesia. Despite these advantages, many EM trainees do not receive focused education in UGRA and there is no published curriculum specifically for EM physicians. The objective of this study was to identify the components of a UGRA curriculum for EM physicians. Methods A list of potential curriculum elements was developed through an extensive literature review. An expert panel was convened that included 13 ultrasound faculty members from 12 institutions and from a variety of practice environments and diverse geographical regions. The panel voted on curriculum elements through two rounds of a modified Delphi process. Results The panelists voted on 178 total elements, 110 background knowledge elements, and 68 individual UGRA techniques. A high level of agreement was achieved for 65 background knowledge elements from the categories: benefits to providers and patients, indications, contraindications, risks, ultrasound skills, procedural skills, sterile technique, local anesthetics, and educational resources. Ten UGRA techniques achieved consensus: interscalene brachial plexus, supraclavicular brachial plexus, radial nerve, median nerve, ulnar nerve, serratus anterior plane, fascia iliaca, femoral nerve, popliteal sciatic nerve, and posterior tibial nerve blocks. Conclusions The defined curriculum represents ultrasound expert opinion on a curriculum for training practicing EM physicians. This curriculum can be used to guide the development and implementation of more robust UGRA education for both residents and independent providers.

Different fertiliser products are commonly promoted for use on pastures in order to improve pasture productivity and support a more ‘healthy’ soil microbial environment. However, minimal field research has been conducted to validate such claims. A 6-year study (2009–14) was conducted on phosphorus (P)-deficient soils at three sites near Yass, New South Wales, to investigate the effect of topdressing perennial native-based pastures with a range of alternative fertilisers compared with single superphosphate and an unfertilised control treatment. The alternative fertiliser products included manures, composts, crushed rock, rock-phosphate-derived products, concentrated ash and microbial products. Annual measurements were made of soil chemical properties, botanical composition and pasture yield during spring and/or winter + spring, as well as the relative effectiveness of products per unit of pasture grown. Soil microbial community structure under each fertiliser treatment was also analysed in the sixth year of the study. Fertiliser products with substantial quantities of P increased extractable soil P and resulted in significantly higher pasture growth and clover content compared with the unfertilised control. Superphosphate was found to be the most P-effective fertiliser for increasing pasture growth, along with a range of other products that showed differential responses. However, the cost and P-effectiveness of the products in relation to pasture growth varied considerably and was a function of rate and frequency of application as well as amount and solubility of the P applied. Despite large differences in pasture growth across the various fertiliser treatments, there was no significant effect of the alternative fertiliser products on microbial community structure compared with either the superphosphate or unfertilised control treatments. The observed variation in bacterial, fungal and archaeal community structures across all fertiliser treatments was best explained by soil pH or aluminium (Al) concentration, which was influenced differentially by the fertiliser products. Fungal community structure was also correlated with pasture-productivity parameters (i.e. spring pasture yield, clover content and soil-available P). Our findings reveal a highly resilient soil microbial community that was influenced minimally by use of the alternative fertiliser products, thus highlighting that on-farm management decisions regarding fertiliser product choice should primarily focus on pasture response and cost-effectiveness.

Paleoclimate proxy evidence suggests a southward shift of the Intertropical Convergence Zone (ITCZ) during times of Northern Hemisphere cooling, including the Last Glacial Maximum, 19–23 ka before present. However, evidence for movement over the Pacific has mainly been limited to precipitation reconstructions near the continents, and the position of the Pacific marine ITCZ is less well constrained. In this study, we address this problem by taking advantage of the fact that the upper ocean density structure reflects the overlying wind field. We reconstruct changes in the upper ocean density structure during the LGM using oxygen isotope measurements on the planktonic foraminifera G. ruber and G. tumida in a transect of sediment cores from the Western Tropical Pacific. The data suggests a ridge in the thermocline just north of the present-day ITCZ persists for at least part of the LGM, and a structure in the Southern Hemisphere that differs from today. The reconstructed structure is consistent with that produced in a General Circulation Model with both a Northern and Southern Hemisphere ITCZ.

While conservation management is increasingly turning towards an ecosystem-level framework, the focus on a small subset of surrogate species has recognised merit given insufficient time, resources, and expertise. The kaka ( Nestor meridionalis ), a large threatened New Zealand parrot, is an iconic, visible species in lowland forests. As kaka populations are sensitive to mustelid predation and habitat loss, kaka can act as both a flagship and indicator species for healthy lowland forest ecosystems in New Zealand. To ensure the sustained protection of kaka over a sufficient area, this research aims to estimate the minimum viable population (MVP) size of kaka in the Eglinton Valley, Fiordland, and the management required for population persistence. A post-breeding census, stochastic, age structured Leslie matrix model was developed to estimate the population size having a 95% probability of persistence over 100 years. Scenarios modeling current and alternate management regimes, uncertain life-history traits, and environmental unpredictability were run. The most ‘realistic’ scenario resulted in an MVP size of 258 kaka (155 adults). Maintaining current levels of predator control appears essential to ensure kaka population persistence. An area of >500 km 2 is proposed to maintain the MVP of kaka based on detailed information on home range size and territory overlap derived from radio-tracking studies. As one of a group of surrogate species in lowland forest ecosystems, kaka may be used to guide management decisions regarding large-scale mustelid trapping and the delineation of habitat area requiring protection in the face of proposed human developments in the region.

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Rifleman, or titipounamu Acanthisitta chloris, is New Zealand’s smallest endemic passerine. The species has a fragmented distribution and is threatened in the Rakiura region in the south of the South Island. The only known population of South Island rifleman A. c. chloris in the Rakiura region persisted on Codfish Island/Whenua Hou. To create a second population of rifleman in Rakiura, 30 caught from Codfish Island were reintroduced onto nearby Ulva Island in February 2003, the first translocation of rifleman. Survival and dispersal were monitored for 1 month post-release, and subsequently during the first and second breeding seasons. Mortality was greatest during holding and transfer, with low to moderate post-release mortality. All founding pairs bred in the first breeding season, and both founders and offspring bred in the second season. Dispersal across the island was greater for offspring. A simple deterministic matrix model indicated positive annual population growth (λ = 1.33), and low risk of short-term extinction. Holding/transfer techniques should be improved for future reintroductions, and longer-term monitoring should be undertaken for a more accurate assessment of vital rates. Based on the survival of founding birds, reproduction by the release generation and their offspring, and high probability of population persistence, the rifleman reintroduction was considered to be successful and a good model for future reintroductions of small passerine birds.

Summary Removing API dope from the makeup process of threaded premium connections during casing and tubing operations not only reduces the risk of contamination of the local environment but also provides a cleaner and safer working area. Conversely, the removal of the antigalling property inherent in API dope greatly increases the risk of galling damage to connections during power-tight makeup. This paper describes various tests carried out to evaluate manufacturers' claims of having successfully improved the galling resistance of premium connections by electro-depositing a white bronze (45%Cu-55%Sn) alloy onto couplings manufactured from high-chrome materials. Galling resistance was measured by subjecting several test coupons plated with copper and various alloy compositions to a series of tests designed to generate high stresses to threaded surfaces. The paper also shows through experimentation how the correct amount of preload was achieved in a premium connection's gas-tight seal by applying a thin lubricating layer (also referred to as a protective film) to both pin and box threads.

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual “swarming” phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa .

A bioassay was developed to identify stimuli that promote the transcriptional induction of the algD operon for alginate biosynthesis in Pseudomonas aeruginosa. Strain PAO1 carried the algD promoter fused to a chloramphenicol acetyl-transferase cartridge (PalgD-cat), and > 50 compounds were tested for promoting chloramphenicol resistance. Most compounds showing PalgD-cat induction were cell wall-active antibiotics that blocked peptidoglycan synthesis. PalgD-cat induction was blocked by mutations in the genes for sigma22 (algT/algU) or regulators AlgB and AlgR. Anti-sigma factor MucA was the primary regulator of sigma22 activity. A transcriptome analysis using microarrays verified that the algD operon undergoes high induction by D-cycloserine. A similar sigma(E)-RseAB complex in Escherichia coli responds to envelope stress, which requires DegS protease in a regulated intramembrane proteolysis (RIP) cascade to derepress the sigma. Mutant phenotypic studies in P. aeruginosa showed that AlgW (PA4446) is likely to be the DegS functional homologue. A mutation in algW resulted in a complete lack of PalgD-cat induction by D-cycloserine. Overexpression of algW in PAO1 resulted in a mucoid phenotype and alginate production, even in the absence of cell wall stress, suggesting that AlgW protease plays a role in sigma22 activation. In addition, a mutation in gene PA3257 (prc), encoding a Prc-like protease, resulted in poor induction of PalgD-cat by D-cycloserine, suggesting that it also plays a role in the response to cell wall stress.

We describe the synthesis of two cyclobutadiene(cyclopentadienyl)cobalt-containing poly(p-phenylene ethynylene)s (PPEs) and their use as precursors for stable ceramic surface coatings. Organometallic PPEs were shaped into hexagonally ordered assemblies by using the breath-figure method. Such breath figures can be washed away with an appropriate solvent. Upon pyrolysis at 500 degrees C under either nitrogen or air, the bubble arrays persist as ceramics and are insoluble in organic solvents or water. The formed pyrolyzed bubble arrays were analyzed by optical and scanning electron microscopy, as well as energy dispersive X-ray microanalysis (EDX). The composition of the ceramic materials is discussed based on EDX and IR data.

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P

In vitro detection of T-cell responses to autoantigens in type 1 diabetes is recognized as being technically challenging. We aimed to accurately measure cellular responses to proinsulin in patients with diabetes, and speculated that presentation of antigen by dendritic cells (DCs) would enhance the sensitivity of the peripheral blood assay. Antigen was mannosylated to facilitate uptake through DC surface mannose receptors to further improve the assay. Whole proinsulin, as well as mannosylated peptides of proinsulin, were combined with peripheral T cells and autologous immature DCs in a proliferative assay in a panel of newly diagnosed type 1 diabetic patients. The DC-based assay detected responses to proinsulin in five of 15 diabetic patients compared to one of 15 diabetic patients detected using the standard mononuclear cell assay. When the results of all patients were combined, the DC assay, but not the mononuclear cell assay, had a proinsulin response that was significantly higher than background (P < 0·001). The DC assay was, however, associated with high autologous mixed lymphocyte reactions that possibly masked responses in individual patients. Mannosylated antigen was taken up in larger quantities than non-mannosylated antigen, but not presented any more powerfully. Our data suggest that autologous DC-based assays are more powerful than standard peripheral blood mononuclear cell assays. However, they are compromised by high autologous mixed lymphocyte reactions and this requires addressing before they can be used as a routine readout of in vitro peripheral T-cell responses.

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterized by immunity against pancreatic islet-derived proteins. The object of this study was to measure antibody and T-cell responses against proinsulin (PI), an islet-derived protein, and to map its dominant T-cell epitopes. Methods: Antibody responses to proinsulin, insulin, glutamic acid decarboxylase (GAD), protein tyrosine phosphatase IA-2 and islet-cell antigen were measured in 116 newly diagnosed diabetic subjects aged 16 to 40 years. T-cell proliferative responses to proinsulin and proinsulin peptides were measured in 33 of these diabetic subjects and in 21 healthy control subjects. Results: 22% of diabetic subjects but no control subjects expressed antibodies to proinsulin. A strong correlation existed between antibody levels to proinsulin and insulin within diabetic subjects. Similar proportions of diabetic (12%) and healthy (9.5%) subjects displayed T-cell responses to proinsulin. There was no correlation between antibody and T-cell responses to proinsulin within subjects. Amino acid region 56 to 72 was identified as the major T-cell epitope of proinsulin, though significant responses to region 14 to 37 were also present. Conclusion: Elevated proinsulin autoantibodies in diabetic subjects confirm proinsulin is an important autoantigen in type 1 diabetes. Though elevated cellular immunity to proinsulin protein was not detected, two dominant T-cell epitopes of proinsulin were identified that span the C-peptide and insulin junctions. Immunity to proinsulin was lower than that reported for childhood-onset type 1 diabetes and we propose that, like insulin, proinsulin may be targeted less frequently in adulthood.

The cDNAs from Hyoscyamus niger , encoding two enzymes of tropane-alkaloid biosynthesis, tropinone reductase I (TR-I) and hyoscyamine-6b-hydroxylase (H6H), have been simultaneously introduced into Nicotiana tabacum using particle bombardment and expressed under the control of the CaMV 35S promoter. Southern and Northern analyses confirmed integration and transcript accumulation for both tr1 and h6h . Fertile tobacco plants expressing both transgenes were regenerated and detached leaves of these plants were fed with tropinone and hyoscyamine, the substrates, respectively, of TR-I and H6H. Besides the expected TR-I and H6H reaction products, acetylated forms of tropine were also generated in these experiments, indicating that the expression of alkaloidpathway enzymes in a transgenic background can produce unexpected substances. In addition, leaves of the transgenic plants showed in most cases higher nicotine content than leaves of control plants. Nicotine levels were approximately 3- to 13-fold higher in both the parental transgenic lines and in T1 progeny expressing functional TR-I and H6H, compared to levels in wild-type plants and in transgenic plants carrying the nptII transgene alone. In addition, nicotine related compounds such as anatabine, nornicotine, bipyridine, anabasine, and myosmine were identified in transgenic tobacco lines and below detection limit in wild-type plants, suggesting changes in the activity of the enzymes in the nicotine biosynthetic pathway in the transgenic background. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

OBJECTIVE—Almost 90% of type 1 diabetes appears in individuals without a close family history. We sought to evaluate the best current predictive strategy, multiple defined autoantibodies, in a long-term prospective study in the general population.RESEARCH DESIGN AND METHODS—Autoantibodies to pancreatic islets (islet cell antibodies [ICAs]) and defined autoantibodies (d-aab) to human GAD, IA2/ICA512, and insulin were tested in 4,505 Washington schoolchildren. Eight years later, 3,000 (67%) subjects were recontacted, including 97% of subjects with any test >99th percentile.RESULTS—Six subjects developed diabetes (median interval 2.8 years), all from among the 12 individuals with multiple d-aab, representing 50% positive predictive value (95% CI 25–75%) and 100% sensitivity (58–100%). Among the others, diabetes occurred in 0 of 6 with one d-aab plus ICA, 0 of 26 with ICA only, 0 of 7 with one d-aab equaling the 99th percentile and another d-aab equaling the 97.5th percentile, 0 of 86 with one d-aab, and 0 of 2,863 with no d-aab or ICA. Adjusted for verification bias, multiple d-aab were 99.9% specific (99.86–99.93%). At this age, new d-aab seldom appeared. Once present, d-aab usually persisted regardless of disease progression, although less so for insulin autoantibodies. Insulin secretion by sequential glucose tolerance testing remained normal in four multiple d-aab subjects not developing diabetes. Of children developing diabetes, five of six (83%) would be included if HLA-DQ genotyping preceded antibody testing, but HLA-DQ did not explain outcomes among high-risk subjects, even when considered along with other genetic markers.CONCLUSIONS—Multiple d-aab were established by age 14 years and prospectively identified all schoolchildren who developed type 1 diabetes within 8 years.

We describe an efficient process for the regeneration of Lonicera tatarica plants from cultured stem sections. Induction of multiple shoots was achieved directly from cultured stem cuttings. The highest regeneration rate was achieved an Gamborg's B5 medium supplemented with 4% sucrose and 0.8% Difco bacto-agar in the absence of hormones. Differentiated shoots were elongated for 5-7 days an induction medium supplemented with 0.5 mg/l GA3. Shoot induction and elongation experiments were carried out using original stem explants from either 2-, 6-, or 18-month-old donor plants. The age of the donor plant had no noticeable effect on either process. However, rooting of elongated shoots occurred only with shoots derived from 2-month-old donor plants. Rooting efficiency and proliferation were highest on half-strength WPM medium supplemented with 2 µM indole-3-butyric acid and 0.6% Keylis agar. The plants regenerated from stem explants were morphologically normal, and levels of loganin and secologanin were comparable to those detected in plants grown from seed and maintained through vegetative propagation.

We have developed a 330-370GHz SIS mixer for small-format, heterodyne, astronomical imaging arrays. Fixed-tuned broadband operation is achieved by means of a superconducting radial waveguide probe. A horn-reflector antenna provides high-efficiency optical coupling. Using a variable-temperature cryogenic noise source, we measured a DSB system noise temperature of 32±1K. The mixer contributes 3±3K, supporting the theoretically-predicted result that the noise temperature of a DSB mixer can be less than hω/2κ (8.6K)

A linear correlation between the ratio of the[CII( $$^{\text{2}}$$ P $$_{3/2}$$ → $$^{\text{2}}$$ P $$_{1/2}$$ )] line intensity to the [ $$^{{\text{12}}}$$ CO(J:1 →0)] line emission, I $$_{{\text{CII}}}$$ /I $$_{{\text{CO}}}$$ and the $${\text{H}}_\alpha$$ equivalent width (EW) is found, over the range 2–71 A in $${\text{H}}_\alpha$$ EW, for a sample of 21late-Type= galaxies. The latter is comprised of an optically selected sample of 12 normal Virgo Cluster spiral galaxies with [CII] detections obtained by us with ISOLWS, plus nine late-Type= galaxies with higher star formation rates (SFRs), for which [CII] data and, especially, $${\text{H}}_\alpha$$ EW data are available in the literature. As a result we infer I $$_{{\text{CII}}}$$ /I $$_{{\text{CO}}}$$ to be a reliable tracer of the current mass-normalized global SFR for non-starburst spiral galaxies. Moreover, the ratio of the [CII] line to the total far-infrared (FIR) continuum intensity, I $$_{{\text{CII}}}$$ /I $$_{{\text{FIR}}}$$ , is found to decrease from ∼0.5% to ∼0.1% with decreasing SFR which we propose is due to a `[CII]-quiet' component of I $$_{{\text{FIR}}}$$ from dust heated by the general interstellar radiation field (ISRF). The more `quiescent' galaxies in the sample have values of I $$_{{\text{CII}}}$$ /I $$_{{\text{CO}}}$$ different from those observed in `compact' Galactic interstellar regions. Their [CII]-emission is interpreted to be dominated by diffuse regions of the interstellar medium (ISM). For normal `star-forming' galaxies the diffuse component of the [CII] emission is estimated to account for at least 50% of the total.

Transposon mutagenesis was used to identify a new locus required for twitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same KpnI restriction fragment at approximately 40 min on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 2·8 kb ORF, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The P. aeruginosa genome also possesses a paralogue of fimV. Homologues of fimV were also found in the sequenced genomes of the other type-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila and Vibrio cholerae, but not in those of other bacteria which lack type IV fimbriae. A fimV homologue was also found in the genome sequence of Shewanella putrefaciens, along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to fimV mutants by complementation in a dosage-dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.

Two cDNA clones were isolated from pea (Pisum sativum L.) and their deduced amino acid sequences shown to have significant homology to phosphoglucomutases from eukaryotic and prokaryotic sources. The longer cDNA contained a putative transit-peptide-encoding sequence, supporting the hypothesis that the isolated clones represent the cytosolic and plastidial isoforms of phosphoglucomutase in pea. Plastid protein import assays confirmed that the putative plastidial isoform was targeted to the plastid stroma where it was proteolytically processed. Expression, co-segregation, linkage, and molecular analyses have confirmed that therug3 locus of pea encodes plastidial phosphoglucomutase. Mutations at this locus result in a near-starchless phenotype of the plant.

We report an efficient whole plant transformation system for Hyoscyamus muticus, an important medicinal plant of the Solanaceous family. We developed a system using a plasmid carrying the nptII and gusA genes, which was delivered into leaf explants by particle bombardment. Ten percent of bombarded leaf explants formed kanamycin-resistant callus, from which putative transgenic plants were recovered. The nptII gene conferring kanamycin resistance was found to be incorporated into the genome of all transgenic plants screened. Over 50% of the kanamycin resistant plants showed strong expression of the non-selected gusA gene. The majority of transgenic plants reached maturity, could be self pollinated, and produced fertile seed. A simple and efficient whole plant transformation system for this medicinal plant is an important step in furthering our understanding of tropane alkaloid production in plants.

A linear correlation between the ratio of the[CII(\(^{\text{2}}\)P\(_{3/2}\)→\(^{\text{2}}\)P\(_{1/2}\))] line intensity to the [\(^{{\text{12}}}\)CO(J:1 →0)] line emission, I\(_{{\text{CII}}}\)/I\(_{{\text{CO}}}\) and the \({\text{H}}_\alpha\) equivalent width (EW) is found, over the range 2–71 A in \({\text{H}}_\alpha\) EW, for a sample of 21late-Type= galaxies. The latter is comprised of an optically selected sample of 12 normal Virgo Cluster spiral galaxies with [CII] detections obtained by us with ISOLWS, plus nine late-Type= galaxies with higher star formation rates (SFRs), for which [CII] data and, especially,\({\text{H}}_\alpha\) EW data are available in the literature. As a result we infer I\(_{{\text{CII}}}\)/I\(_{{\text{CO}}}\) to be a reliable tracer of the current mass-normalized global SFR for non-starburst spiral galaxies. Moreover, the ratio of the [CII] line to the total far-infrared (FIR) continuum intensity, I\(_{{\text{CII}}}\)/I\(_{{\text{FIR}}}\), is found to decrease from ∼0.5% to ∼0.1% with decreasing SFR which we propose is due to a `[CII]-quiet' component of I\(_{{\text{FIR}}}\) from dust heated by the general interstellar radiation field (ISRF). The more `quiescent' galaxies in the sample have values of I\(_{{\text{CII}}}\)/I\(_{{\text{CO}}}\) different from those observed in `compact' Galactic interstellar regions. Their [CII]-emission is interpreted to be dominated by diffuse regions of the interstellar medium (ISM). For normal `star-forming' galaxies the diffuse component of the [CII] emission is estimated to account for at least 50% of the total.

We have developed an efficient direct DNA transfer procedure for the facile engineering of Catharanthus roseus cell cultures. Particle bombardment of callus derived from leaf material permitted rapid selection and establishment of transgenic cell lines. Transgenic callus were recovered at a frequency of between 60–80% of total callus bombarded with a single plasmid. Bombardment using two separate plasmids resulted in a 25–60% frequency of transgenic callus recovered, up to 90% containing both input plasmids. Between 10–20 g FW of transgenic material was produced within 3 months of bombardment, providing sufficient material for molecular and biochemical analyses. We developed two complementary systems allowing selection on either hygromycin or kanamycin to permit re-transformation using plasmids carrying additional genes of interest. Use of leaf tissue as explant for transformation avoids time-consuming and labor intensive procedures involving suspension cultures. We provide molecular data on integration and expression of selected and non selected transgenes in a number of transgenic callus lines. Transgene integration events for co-transformed plasmids were relatively simple, occurring at one or two sites in the genome for most of the lines we analysed. Molecular analysis of callus resulting from co-transformation experiments using two different plasmids revealed that in nine of 10 putative transgenic lines we selected for analysis both plasmids had integrated into the genome. RNA gel-blot analysis and histochemical staining showed that an unselected transgene, gusA, was expressed in seven of the ten lines we analysed.

Intermittent, low levels of pesticide in the borehole water at Debden Road water works, Saffron Walden, England, required an appropriate treatment for its removal. A second treatment goal was a reduction of the calcium hardness by about 50%. Nanofiltration utilising DOW NF 200 was the chosen process allowing to achieve both requirements. During the first year of operation, no significant decrease in permeability was observed and the plant was cleaned only once after an incident that caused heavy scaling on the membranes of the third stage. This scaling event was also clearly visible in the monitoring graph of the differential pressure normalized for the tangential flow rate. The pesticide concentrations in the permeate always remained far below the maximum values of drinking water standards. Due to a severe drought in the area, the plant had to be run at a feed flow rate that was 35% below its design capacity. The consequence of the low flux rate was an increased calcium passage that resulted in a calcium hardness of the permeate above the desired maximum. Several solutions were proposed to increase the calcium rejection and the chosen measure was to take 7 vessels out of service until the water availability will allow running the plant at its design capacity.

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium , the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.

We have created a population of transgenic tobacco plants carrying cDNAs encoding two consecutive enzymes from early stages in monoterpenoid alkaloid biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan decarboxylase (tdc) and strictosidine synthase (str1) together with a selectable marker gene, were introduced on a single transforming plasmid into tobacco leaves by particle bombardment. Analysis of 150 independent transgenic plants at the DNA and RNA levels demonstrated a range of integration events and steady-state transcript levels for the tdc and str1 transgenes. Southern blot analysis indicated that the tdc and str1 transgenes were integrated at least once in all 150 transformants giving a 100% co-integration frequency of the two unselected genes carried on the same plasmid. A comparison of Southern and northern data suggested that in 26% of the plants, both tdc and str1 transgenes were silenced, 41% demonstrated a preferential silencing of either the tdc or the str1 transgene, with the remaining 33% of the plants expressing both transgenes. We observed no clear correlation between the number of integration events of a specific transgene and the levels of accumulated transcript. Twenty plants representing the range of molecular diversity in the transgenic population were selected for further analysis. Seeds were collected from self-fertilised transformants and germinated on medium containing kanamycin. Seedlings were harvested after 7 weeks and TDC and STR1 enzymatic assays were carried out. We observed a 24- and 110-fold variation in levels of TDC and STR1 activities, respectively. Our data correlate molecular diversity with biochemistry and accumulation of end-product and provide a detailed molecular and biochemical characterization of transgenic plants transformed with a single plasmid carrying two genes of secondary metabolism.

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.

Obesity and insulin resistance have been linked to rising incidence and earlier onset of Type 1 diabetes. Inherited differences in insulin action might also influence the evolution of Type 1 diabetes.Our aim was to determine whether parental BMI and insulin resistance influences age of onset of Type 1 diabetes in their offspring.BMI standard deviation score and age at diagnosis of Type 1 diabetes was examined in 227 children, and in 206 of these was compared with local matched control subjects. Non-diabetic parents of a subgroup of 80 children with Type 1 diabetes were recruited. Parental BMI was compared with local adult control subjects. The relationship between parental BMI, waist-hip ratio, homeostasis model assessment of insulin resistance (HOMA-IR), leptin and adiponectin levels and age at diagnosis of Type 1 diabetes in offspring was examined.We found no relationship between age at diagnosis of Type 1 diabetes in children and BMI standard deviation score (P = 0.5). Children with Type 1 diabetes and their parents were heavier than matched control subjects (mean BMI standard deviation score sd in children = 0.66 1.06 vs. 0.32 1.16 in control subjects, P = 0.002; mean parental BMI sd 27.7 0.4 vs. 25.5 0.4 kg ⁄m2 in control subjects; P0.0001). Maternal HOMA-IR accounted for 20% of variation in age at diagnosis (P0.001) with increasing maternal insulin resistance associated with later age at diagnosis of Type 1 diabetes.Childrenwith Type 1 diabetes and their parents have an increased BMI at diagnosis.Maternal insulin resistance is associated with later onset of Type 1 diabetes in children.