Coarse spectral characterization of warm x-rays at the Z facility using a filtered thermoluminescent dosimeter array.

A new collimated filtered thermoluminescent dosimeter (TLD) array has been developed at the Z facility to characterize warm x-rays (hν > 10 keV) produced by Z pinch radiation sources. This array includes a Kapton debris shield assembly to protect the TLDs from the source debris, a collimator array to limit the field of view of the TLDs to the source region, a filter wheel containing filters of aluminum, copper and tungsten up to 3 mm thick to independently filter each TLD, and a hermetically sealed cassette containing the TLDs as well as tungsten shielding on the sides and back of the array to minimize scattered radiation reaching the TLDs. Experimental results from a krypton gas puff and silver wire array shot are analyzed using two different functional forms of the energy spectrum to demonstrate the ability of this diagnostic to consistently extend the upper end of the x-ray spectrum characterization from ∼50 keV to >1 MeV.

Testing small molecule analogues of the Acanthocheilonema viteae immunomodulator ES-62 against clinically relevant allergens.

ES-62 is a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that protects against ovalbumin (OVA)-induced airway hyper-responsiveness in mice by virtue of covalently attached anti-inflammatory phosphorylcholine (PC) residues. We have recently generated a library of small molecule analogues (SMAs) of ES-62 based around its active PC moiety as a starting point in novel drug development for asthma and identified two compounds - termed 11a and 12b - that mirror ES-62's protective effects. In this study, we have moved away from OVA, a model allergen, to test the SMAs against two clinically relevant allergens - house dust mite (HDM) and cockroach allergen (CR) extract. We show that both SMAs offer some protection against development of lung allergic responses to CR, in particular reducing eosinophil infiltration, whereas only SMA 12b is effective in protecting against eosinophil-dependent HDM-induced allergy. These data therefore suggest that helminth molecule-induced protection against model allergens may not necessarily translate to clinically relevant allergens. Nevertheless, in this study, we have managed to demonstrate that it is possible to produce synthetic drug-like molecules based on a parasitic worm product that show therapeutic potential with respect to asthma resulting from known triggers in humans.

Measurements of high-current electron beams from X pinches and wire array Z pinches.

Some issues concerning high-current electron beam transport from the X pinch cross point to the diagnostic system and measurements of the beam current by Faraday cups are discussed. Results of computer simulation of electron beam propagation from the pinch to the Faraday cup give limits for the measured current for beams having different energy spreads. The beam is partially neutralized as it propagates from the X pinch to a diagnostic system, but within a Faraday cup diagnostic, space charge effects can be very important. Experimental results show evidence of such effects.

Novel quorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium.

Seven new genes controlled by the quorum-sensing signal molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) have been identified in Erwinia carotovora subsp. carotovora. Using TnphoA as a mutagen, we enriched for mutants defective in proteins that could play a role in the interaction between E. carotovora subsp. carotovora and its plant hosts, and identified NipEcc and its counterpart in E. carotovora subsp. atroseptica. These are members of a growing family of proteins related to Nep1 from Fusarium oxysporum which can induce necrotic responses in a variety of dicotyledonous plants. NipEcc produced necrosis in tobacco, NipEca affected potato stem rot, and both affected virulence in potato tubers. In E. carotovora subsp. carotovora, nip was shown to be subject to weak repression by the LuxR family regulator, EccR, and may be regulated by the negative global regulator RsmA.

PECAM-1 interacts with nitric oxide synthase in human endothelial cells: implication for flow-induced nitric oxide synthase activation.

OBJECTIVE: We have previously shown that fluid shear stress (FSS) triggers endothelial nitric oxide synthase (eNOS) activity in endothelial cells and that the mechanotransduction mechanisms responsible for activation discriminate between rapid changes in FSS and FSS per se. We hypothesized that the particular sublocalization of eNOS at the cell-cell junction would render it responsive to activation by FSS temporal gradients. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), immunofluorescence revealed strong eNOS membrane staining at the cell-cell junction colocalizing with platelet/endothelial cell adhesion molecule-1 (PECAM-1). In PECAM-1-/- mouse aorta, eNOS junctional localization seen in the wild type was absent. Similarly, junctional staining was lost in wild-type aorta near intercostal artery branches. eNOS/PECAM-1 association in HUVECs was confirmed by coimmunoprecipitation. When HUVECs were subjected to a 0.5s impulse of 12 dynes/cm2, a transient disruption of the eNOS/PECAM-1 complex was observed, accompanied by an increase in eNOS activity (cGMP production). Ramped flow did not trigger complex dissociation or an increase in cGMP production. In a cell-free system, a direct inhibition of eNOS activity by PECAM-1 is shown. CONCLUSIONS: These results suggest that eNOS is complexed with PECAM-1 at the cell-cell junction and is likely involved in the modulation of eNOS activity by FSS temporal gradients but not by FSS itself.

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Acute reversal of lipid-induced muscle insulin resistance is associated with rapid alteration in PKC-theta localization.

Muscle insulin resistance in the chronic high-fat-fed rat is associated with increased membrane translocation and activation of the novel, lipid-responsive, protein kinase C (nPKC) isozymes PKC-theta and -epsilon. Surprisingly, fat-induced insulin resistance can be readily reversed by one high-glucose low-fat meal, but the underlying mechanism is unclear. Here, we have used this model to determine whether changes in the translocation of PKC-theta and -epsilon are associated with the acute reversal of insulin resistance. We measured cytosol and particulate PKC-alpha and nPKC-theta and -epsilon in muscle in control chow-fed Wistar rats (C) and 3-wk high-fat-fed rats with (HF-G) or without (HF-F) a single high-glucose meal. PKC-theta and -epsilon were translocated to the membrane in muscle of insulin-resistant HF-F rats. However, only membrane PKC-theta was reduced to the level of chow-fed controls when insulin resistance was reversed in HF-G rats [% PKC-theta at membrane, 23.0 +/- 4.4% (C); 39.7 +/- 3.4% (HF-F, P < 0.01 vs. C); 22.5 +/- 2.7% (HF-G, P < 0.01 vs. HF-F), by ANOVA]. We conclude that, although muscle localization of both PKC-epsilon and PKC-theta are influenced by chronic dietary lipid oversupply, PKC-epsilon and PKC-theta localization are differentially influenced by acute withdrawal of dietary lipid. These results provide further support for an association between PKC-theta muscle cellular localization and lipid-induced muscle insulin resistance and stress the labile nature of high-fat diet-induced insulin resistance in the rat.

Depletion of glutathione and ascorbate in lung lining fluid by respirable fibres.

OBJECTIVE: The use of synthetic vitreous fibres has increased along with a decline in the utilisation of asbestos. There remains concern that these synthetic fibres pose a health risk to workers because of the generation of respirable fibres which can enter the lung and cause adverse health effects. An improved understanding of the mechanism of fibre pathogenicity should allow more rational short-term testing regimes for new fibres as they are developed. We hypothesised that carcinogenic fibres have greater free radical activity compared with non-carcinogenic fibres and that they contribute to disease by causing oxidative stress in the lung. We examined a panel of respirable fibres, designated as being carcinogenic or non-carcinogenic based on previous animal studies for ability to deplete antioxidants from lung lining fluid. METHODS: On the basis of inhalation studies, a panel of fibres was divided into three carcinogenic fibres-amosite asbestos, silicon carbide, and refractory ceramic fibre 1 (RCF1) and three non-carcinogenic fibres-man-made vitreous fibre 10 (a glass fibre MMVF10), Code 100/475 glass fibre, and refractory ceramic fibre 4 (RCF4). We measured the levels of glutathione (GSH) and ascorbate, two antioxidants present in lung lining fluid (LLF) after fibre treatment. All of the experiments were carried out at equal fibre number. RESULTS: Fibres had the ability to deplete both GSH and ascorbate from both LLF and pure solutions, an effect which was fibre number dependent. The greatest depletion of antioxidants was observed with the two non-carcinogenic glass fibres, and this effect was observed when A549 lung epithelial cells were treated with fibres. CONCLUSIONS: Our results show that antioxidant depletion in cell free solution and lung lining fluid solely is not a simple indicator of the ability of fibres to cause lung pathology and that other biological events in the lung are involved.

Cloning and expression of a DAX1 homologue in the chicken embryo.

DAX1 is an unusual member of the orphan nuclear receptor family of transcription factors. Mutations in human DAX1 cause X-linked adrenal hypoplasia congenita, while abnormal duplication of the gene is responsible for male-to-female dosage-sensitive sex reversal. Based on these and other observations, DAX1 is thought to play a role in adrenal and gonadal development in mammals. As DAX1 has not previously been described in any other vertebrate, a putative avian DAX1 clone was isolated from an embryonic chicken (Gallus domesticus) urogenital ridge cDNA library. The expression profile of this cDNA was then examined during gonadogenesis. The clone included the conserved 3' ligand-binding motif identified in humans and mice but the 5' region lacked the repeat motif thought to specify a DNA-binding domain in mammals. Southern blot analysis and fluorescence in situ hybridisation mapping showed that the gene is autosomal, located on chromosome 1q. Sequence comparisons showed that the putative chicken DAX1 protein has 63 and 60% identity with the human and mouse proteins respectively over the region of the conserved ligand-binding domain. However, stronger identity (74%) exists with a putative alligator DAX1 sequence over the same region. Northern blotting detected a single 1.4 kb transcript in late embryonic chicken gonads, while RNase protection assays revealed expression in the embryonic gonads of both sexes during the period of sexual differentiation. Expression increased in both sexes during gonadogenesis, but was higher in females than in males. This is the first description of a DAX1 homologue in a non-mammalian vertebrate.

Identification and environmental detection of Rhodococcus species by 16S rDNA-targeted PCR.

Bacteria of the genus Rhodococcus can degrade a wide range of organic pollutants and catalyse many useful biotransformations. There is a need for improved tests to identify Rhodococcus species. PCR-based methods for species identification offer advantages in terms of speed and accuracy over traditional methods and can allow direct detection of microbes in environmental samples., PCR tests, using primers targeted at species-specific sequences in the 16S rRNA gene, were successfully developed for R. globerulus, R. erythropolis, R. opacus and R. ruber. These tests gave positive results with all or most strains of target species but did not generally cross-react with other species. Cases of apparent cross-reaction were shown to be due to prior misclassification of strains of R. opacus as R. erythropolis and of strains of R. ruber as R. rhodochrous. A simple and rapid method for the extraction and purification of DNA from soil was developed and successfully applied to the PCR detection of indigenous R. erythropolis in an environmental sample. Cell lysis in the samples was achieved by lysozyme and sarkosyl treatment, aided by freeze-thaw cycles. Removal of humic compounds inhibitory to PCR was accomplished by CTAB treatment with solvent extraction and, if necessary, passage of extracts through Sepharose CL-6B in a spun-column format. Extracts prepared using a tris-EDTA buffer were much clearer than those prepared using a sodium phosphate buffer, indicating lower levels of humic compounds. A detection limit of 104 cfu g-1 of soil was achieved and the use of a secondary PCR allowed detection of 1 cfu g-1.

Diet-induced muscle insulin resistance in rats is ameliorated by acute dietary lipid withdrawal or a single bout of exercise: parallel relationship between insulin stimulation of glucose uptake and suppression of long-chain fatty acyl-CoA.

Chronic high-fat feeding in rats induces profound whole-body insulin resistance, mainly due to effects in oxidative skeletal muscle. The mechanisms of this reaction remain unclear, but local lipid availability has been implicated. The aim of this study was to examine the influence of three short-term physiological manipulations intended to lower muscle lipid availability on insulin sensitivity in high-fat-fed rats. Adult male Wistar rats fed a high-fat diet for 3 weeks were divided into four groups the day before the study: one group was fed the normal daily high-fat meal (FM); another group was fed an isocaloric low-fat high-glucose meal (GM); a third group was fasted overnight (NM); and a fourth group underwent a single bout of exercise (2-h swim), then were fed the normal high-fat meal (EX). In vivo insulin action was assessed using the hyperinsulinemic glucose clamp (plasma insulin 745 pmol/l, glucose 7.2 mmol/l). Prior exercise, a single low-fat meal, or fasting all significantly increased insulin-stimulated glucose utilization, estimated at either the whole-body level (P < 0.01 vs. FM) or in red quadriceps muscle (EX 18.2, GM 28.1, and NM 19.3 vs. FM 12.6 +/- 1.1 micromol x 100 g(-1) x min(-1); P < 0.05), as well as increased insulin suppressibility of muscle total long-chain fatty acyl-CoA (LC-CoA), the metabolically available form of fatty acid (EX 24.0, GM 15.5, and NM 30.6 vs. FM 45.4 nmol/g; P < 0.05). There was a strong inverse correlation between glucose uptake and LC-CoA in red quadriceps during the clamp (r = -0.7, P = 0.001). Muscle triglyceride was significantly reduced by short-term dietary lipid withdrawal (GM -22 and NM -24% vs. FM; P < 0.01), but not prior exercise. We concluded that muscle insulin resistance induced by high-fat feeding is readily ameliorated by three independent, short-term physiological manipulations. The data suggest that insulin resistance is an important factor in the elevated muscle lipid availability induced by chronic high-fat feeding.

Identification of Rhodococcus equi using the polymerase chain reaction.

Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.

Protein kinase C alpha mediates phospholipase D activation by nucleotides and phorbol ester in Madin-Darby canine kidney cells. Stimulation of phospholipase D is independent of activation of polyphosphoinositide-specific phospholipase C and phospholipase A2.

Protein kinase C (PKC) has been implicated in the activation of phospholipase D (PLD) in a number of systems. By antisense technology, we have "knocked out" alpha and beta isoforms of PKC to study the role of these isoforms in PLD activation in Madin-Darby canine kidney (MDCK) cells. To this end, we have studied PLD activation by phorbol 12-myristate 13-acetate (PMA), ATP, UTP, and 2-methylthio-ATP in cells labeled with [3H]palmitic acid. [3H]Phosphatidylethanol (PEt) production catalyzed by PLD in the presence of ethanol was time- and concentration-dependent in PMA- and nucleotide-stimulated cells. In Ca(2+)-free medium, [3H]PEt accumulation was diminished for all stimuli assayed. Treatment of cells with chelerythrine, an inhibitor of PKC, and phorbol ester down-regulation of PKC inhibited [3H]PEt production by both PMA and nucleotides. In cells transfected with antisense PKC alpha or both PKC alpha and PKC beta, PLD activation was inhibited by both PMA and nucleotides, whereas in cells transfected with antisense PKC beta, PLD activation was similar to that of control cells. Moreover, inhibition of polyphosphoinositide-specific PLC (by neomycin) or of release of arachidonic acid and arachidonic acid metabolites (by nordihydroguaiaretic acid or by indomethacin) failed to decrease [3H]PEt accumulation in PMA- and nucleotide-stimulated MDCK-D1 cells. From these data, we conclude that in MDCK-D1 cells PMA and nucleotide receptors utilize PKC alpha to regulate PLD activity and that PLD activation is independent of the activation of polyphosphoinositide-specific PLC and phospholipase A2-mediated release of arachidonic acid or arachidonic acid metabolites.

Inhibition of expression of protein kinase C alpha by antisense cDNA inhibits phorbol ester-mediated arachidonate release.

A major unresolved issue in the area of signal transduction relates to the role of particular isoforms of protein kinase C (PKC) in mediating cellular responses subsequent to activation of that enzyme. We have addressed this issue by the use of antisense technology. We have stably transfected Madin-Darby canine kidney cells with antisense PKC alpha, PKC beta, or both PKC alpha and -beta cDNAs. The transfected cDNA was integrated and expressed. We have isolated cells in which expression of PKC alpha is inhibited. In cells transfected with antisense PKC alpha or both PKC alpha and -beta, phorbol ester-stimulated release of arachidonate and its metabolites was inhibited, whereas in cells transfected with antisense PKC beta cDNA alone, phorbol ester-stimulated arachidonate release was not significantly different from control cells. We thus demonstrate the use of a novel technique to inhibit PKC isoform expression. We show that inhibition of expression of PKC alpha causes a loss in phospholipase A2-mediated arachidonate release. Antisense-inhibited expression of PKC isoforms may provide a useful approach to define additional functions of particular PKC isoforms.

Spectral analysis as a diagnostic aid in the management of high-risk pregnancy.

Spectral analysis of the fetal heart rate and intrauterine pressure was done with data obtained during labor for 24 patients delivered of their infants in an obstetric intensive care unit. The patients were grouped according to clinical assessment of labor and neonatal outcome as normal (11), abnormal/premature (4), and prolonged labor/cesarean section (9). A comparison among groups of spectral density functions and of coherence functions and phase angles failed to reveal consistent features that could be used to distinguish between groups. However, an analysis of the variance (integrated spectrum) and the mean of the base-line component of the fetal heart rate showed that the values for the normal and abnormal/premature groups could be distinguished from the background population. It is suggested that a study of the joint statistics of the variance and the mean may lead to the development of a diagnostic aid to the early detection of incipient fetal distress.