Extended timescale 2D IR probes of proteins: p-cyanoselenophenylalanine.

The importance of dynamics to the function of proteins is well appreciated, but the difficulty in their measurement impedes investigation into their precise role(s). 2D IR spectroscopy is a developing approach for the study of dynamics and has motivated efforts to develop spectrally resolved IR probe groups that enable its application for measuring the dynamics at specific sites in a protein. A challenge with this approach is that the timescales accessible are limited by the vibrational lifetimes of the probes. Toward development of better probes for 2D IR spectroscopy of protein dynamics, we report the characterization of p-cyano-seleno-phenylalanine (CNSePhe), a derivative of the well established IR probe p-cyano-phenylalanine (CNPhe), by FT IR, pump-probe, and 2D IR spectroscopy. The incorporation of the heavy Se atom decouples the CN vibration from the rest in the molecule. Although this leads to a reduction of the transition dipole strength, and thus a reduction in signal intensity, it also dramatically increases the vibrational lifetime, enabling collection of 2D IR spectra for analysis of molecular dynamics on much longer timescales. Interestingly, we also find that the lifetime for CNSePhe shows increased sensitivity to the presence of hydrogen bonding interactions with the CN, suggesting that the probe should be useful for interpretation of CN spectra and possibly for the study of solvation.

Oncolytic reovirus enhances rituximab-mediated antibody-dependent cellular cytotoxicity against chronic lymphocytic leukaemia.

The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo.

Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.

Lymphokine-activated killer and dendritic cell carriage enhances oncolytic reovirus therapy for ovarian cancer by overcoming antibody neutralization in ascites.

Reovirus is an oncolytic virus (OV), which acts by both direct tumor cell killing and priming of antitumor immunity. A major obstacle for effective oncolytic virotherapy is effective delivery of OV to tumor cells. Ovarian cancer is often confined to the peritoneal cavity and therefore i.p. delivery of reovirus may provide the ideal locoregional delivery, avoiding systemic dissemination. However, ovarian cancer is associated with an accumulation of ascitic fluid, which may interfere with oncolytic viral therapy. Here, we investigated the effect of ascites on reovirus-induced oncolysis against primary ovarian cancer cells and ovarian cancer cell lines. In the absence of ascites, reovirus was cytotoxic against ovarian cancer cells; however, cytotoxicity was abrogated in the presence of ascitic fluid. Neutralizing antibodies (NAb) were identified as the cause of this inhibition. Loading OV onto cell carriers may facilitate virus delivery in the presence of NAb and immune cells which have their own antitumor effector activity are particularly appealing. Immature dendritic cells (iDC), Lymphokine-activated killer (LAK) cells and LAKDC cocultures were tested as potential carriers for reovirus for tumor cell killing and immune cell priming. Reovirus-loaded LAKDC, and to a lesser degree iDC, were able to: (i) protect from NAb and hand-off reovirus for tumor cell killing; (ii) induce a proinflammatory cytokine milieu (IFNÉ£, IL-12, IFNα and TNFα) and (iii) generate an innate and specific antitumor adaptive immune response. Hence, LAKDC pulsed with reovirus represent a novel, clinically practical treatment for ovarian cancer to maximise both direct and innate/adaptive immune-mediated tumor cell killing.

Immune activation by combination human lymphokine-activated killer and dendritic cell therapy.

BACKGROUND: Optimal cellular immunotherapy for cancer should ideally harness both the innate and adaptive arms of the immune response. Lymphokine-activated killer cells (LAKs) can trigger early innate killing of tumour targets, whereas long-term adaptive-specific tumour control requires priming of CD8+ cytotoxic lymphocytes (CTLs) following acquisition of tumour-associated antigens (TAAs) by antigen-presenting cells such as dendritic cells (DCs). As DCs stimulate both innate and adaptive effectors, combination cell therapy using LAKs and DCs has the potential to maximise anti-tumour immune priming. METHODS: Reciprocal activation between human clinical grade LAKs and DCs on co-culture, and its immune consequences, was monitored by cell phenotype, cytokine release and priming of both innate and adaptive cytotoxicity against melanoma targets. RESULTS: Co-culture of DCs and LAKs led to phenotypic activation of natural killer (NK) cells within the LAK population, which was associated with increased production of inflammatory cytokines and enhanced innate cytotoxicity against tumour cell targets. The LAKs reciprocally matured DCs, and the combination of LAKs and DCs, on addition of melanoma cells, supported priming of specific anti-tumour CTLs better than DCs alone. CONCLUSION: Clinical-grade LAKs/DCs represents a practical, effective combination cell immunotherapy for stimulation of both innate and adaptive anti-tumour immunity in cancer patients.

Simulated aquatic animal disease outbreaks: a tool for improving responses to emergencies.

Simulated disease outbreaks are vital tools for preparing administrative and functional responses to emergencies. Three aquatic emergency response exercises conducted in Australia are summarised. The prinicipal lesson learned from these exercises was the need for coordination, improved communication and a whole-of-government approach. An enhanced understanding of the legislative instruments required for a response, insights into human factors such as staff rostering, training, counselling and provision of assistance packages were also obtained. Developing a sense of overconfidence about preparedness arrangements (as a result of having tested specific, planned scenarios) must be avoided. Emergencies occur infrequently and erratically; conducting exercises is a vital component of planning processes that ensure that responses to emergency events are effective.

Clonal analysis of invasive pneumococcal isolates in Scotland and coverage of serotypes by the licensed conjugate polysaccharide pneumococcal vaccine: possible implications for UK vaccine policy.

A 7-valent pneumococcal conjugate vaccine (PCV7) has gained licensure and has proven successful in the USA for preventing pneumococcal disease and reducing the incidence of antibiotic-resistant pneumococcal strains. The ability, therefore, to accurately monitor the likely effect of the introduction of PCV7 vaccine on invasive pneumococcal disease in the UK is essential. Serotyping and multilocus sequence typing was performed on invasive isolates of Streptococcus pneumoniae (n=645) from Scotland during 2003. The information gained from this was used to evaluate serotype coverage by the vaccine and the relationship between serotypes. In the present study, invasive pneumococcal disease in Scotland was caused by 33 different serotypes, consisting of 150 sequence types. Overall, 48.4% of the isolates were of serotypes included in the PCV7. Pneumococci were most frequently associated with sequence types 9, 124, and 162. PCV7 would provide protection in 71.8% of infants under 5 years of age against the serotypes in the vaccine. There was limited evidence of the potential for capsule switch among currently circulating invasive pneumococci. The successful implementation of a suitable vaccination programme should lead to a reduction in invasive pneumococcal disease in the UK as well as a reduction in antibiotic resistance of pneumococcal strains.

Erythromycin resistance in invasive serotype 14 pneumococci is highly related to clonal type.

Sixty-seven serotype 14 pneumococci, isolated from invasive disease in Scotland during the first 6 months of 2003, were characterized. Serotype 14 pneumococci accounted for 18.2 % of the total number of cases. Serotyping, multilocus sequence typing and antibiotic susceptibility testing revealed 10 different sequence types (STs), predominantly ST 9 and ST 124; most ST 9 pneumococci were erythromycin-resistant whilst those of ST 124 were not.

Serotypes and sequence types of pneumococci causing invasive disease in Scotland prior to the introduction of pneumococcal conjugate polysaccharide vaccines.

Pneumococcal conjugate polysaccharide (Pnc) vaccines are now available, and the need for an improved understanding of circulating pneumococcal serotypes and sequence types (STs) is recognized. Three hundred sixty-eight pneumococci isolated in cases of invasive disease in Scotland in the first 6 months of 2003 were analyzed. The isolates belonged to 30 serotypes, and there was a strong correlation between serotype and ST, although only nine serotypes consisted of a single ST. The following serotypes coexisted with the following numbers of STs: serotype 14, 10 STs, serotype 8, 8 STs; serotype 4, 6 STs; serotype 22F, 8 STs; serotype 9V, 7 STs; serotype 23F, 6 STs; serotype 6B, 6 STs; serotype 1, 3 STs; serotype 3, 3 STs; and serotype 7F, 3 STs. Our data also showed a strong association between ST and serotype, although 19 STs contained multiple serotypes. Of the 10 most common STs, 6 coexisted with a single serotype each. Vaccine coverage in all age groups was 94.9% for the 23-valent polysaccharide vaccine and 50.7, 55.4, and 64.1% for the 7-, 9-, and 11-valent Pnc vaccines, respectively. For those under the age of 2 years, 79% coverage would be provided by the 7-, 9-, and 11-valent Pnc vaccines.

Importance of the forest canopy to fluxes of methyl mercury and total mercury to boreal ecosystems.

The forest canopy was an important contributor to fluxes of methyl mercury (MeHg) and total mercury (THg) to the forest floor of boreal uplands and wetlands and potentially to downstream lakes, at the Experimental Lakes Area (ELA), northwestern Ontario. The estimated fluxes of MeHg and THg in throughfall plus litterfall below the forest canopy were 2 and 3 times greater than annual fluxes by direct wet deposition of MeHg (0.9 mg of MeHg ha(-1)) and THg (71 mg of THg ha(-1)). Almost all of the increased flux of MeHg and THg under the forest canopy occurred as litterfall (0.14-1.3 mg of MeHg ha(-1) yr(-1) and 110-220 mg of THg ha(-1) yr(-1)). Throughfall added no MeHg and approximately 9 mg of THg ha(-1) yr(-1) to wet deposition at ELA, unlike in other regions of the world where atmospheric deposition was more heavily contaminated. These data suggest that dry deposition of Hg on foliage as an aerosol or reactive gaseous Hg (RGM) species is low at ELA, a finding supported by preliminary measurements of RGM there. Annual total deposition from throughfall and litterfall under a fire-regenerated 19-yr-old jack pine/birch forest was 1.7 mg of MeHg ha(-1) and 200 mg of THg ha(-1). We found that average annual accumulation of MeHg and THg in the surficial litter/fungal layer of soils since the last forest fire varied between 0.6 and 1.6 mg of MeHg ha(-1) and between 130 and 590 mg of THg ha(-1) among sites differing in drainage and soil moisture. When soil Hg accumulation sites were matched with similar sites where litterfall and throughfall were collected, measured fluxes of THg to the forest floor (sources) were similar to our estimates of longterm soil accumulation rates (sinks), suggesting that the Hg in litterfall and throughfall is a new and not a recycled input of Hg to forested ecosystems. However, further research is required to determine the proportion of Hg in litterfall that is being biogeochemically recycled within forest and wetland ecosystems and, thus, does not represent new inputs to the forest ecosystem.

Endobronchial tumors in children: an uncommon clinical entity.

We performed a retrospective chart review of 10 children in whom endobronchial tumors were diagnosed in a tertiary-care children's medical center from 1988 to 1998. Of the 10 patients, 6 were male and 4 were female (8 white, 1 Hispanic, 1 African American). The mean age at presentation was 5.2 years. Eight tumors were benign, and 2 were malignant; 4 were on the right side, 3 were on the left side, and 3 were bilateral. Histologic findings included 3 bronchial papillomas, 3 inflammatory masses, 1 endobronchial hemangioma, 1 leiomyoma, 1 mucoepidermoid carcinoma, and 1 bronchial carcinoid. Endobronchial tumors in children are a rare disorder. The diagnosis requires a high index of suspicion in children with atypical or chronic respiratory complaints. Newer adjuvant medical therapies and surgical innovations offer improved disease control in these patients, and a multidisciplinary approach is often warranted.

Mercury biogeochemistry in the Idrija river, Slovenia, from above the mine into the Gulf of Trieste.

The Idrija Mine is the second largest Hg mine in the world which operated for 500 years. Mercury (Hg)-laden tailings still line the banks, and the system is a threat to the Idrija River and water bodies downstream including the Soca/Isonzo River and the Gulf of Trieste in the northern Adriatic Sea. A multidisciplinary study was conducted in June 1998 on water samples collected throughout the Idrija and Soca River systems and waters and sediments in the Gulf. Total Hg in the Idrija River increased >20-fold downstream of the mine from <3 to >60 ng liter(-1) with methyl mercury (MeHg) accounting for approximately 0.5%. Concentrations increased again downstream and into the estuary with MeHg accounting for nearly 1.5% of the total. While bacteria upstream of the mine did not contain mercury detoxification genes (mer), such genes were detected in bacteria collected downstream. Benthic macroinvertebrate diversity decreased downstream of the mine. Gulf waters near the river mouth contained up to 65 ng liter(-1) total Hg with approximately 0.05 ng liter(-1) MeHg. Gulf sediments near the river mouth contained 40 microgram g(-1) total Hg with MeHg concentrations of about 3 ng g(-1). Hg in sediment pore waters varied between 1 and 8 ng liter(-1), with MeHg accounting for up to 85%. Hg methylation and MeHg demethylation were active in Gulf sediments with highest activities near the surface. MeHg was degraded by an oxidative pathway with >97% C released from MeHg as CO(2). Hg methylation depth profiles resembled profiles of dissolved MeHg. Hg-laden waters still strongly impact the riverine, estuarine, and marine systems. Macroinvertebrates and bacteria in the Idrija River responded to Hg stress, and high Hg levels persist into the Gulf. Increases in total Hg and MeHg in the estuary demonstrate the remobilization of Hg, presumably as HgS dissolution and recycling. Gulf sediments actively produce MeHg, which enters bottom waters and presumably the marine food chain.

Inhibition of inducible nitric oxide synthase by acetamidine derivatives of hetero-substituted lysine and homolysine.

The synthesis and in vitro evaluation of the acetamidine derivatives of hetero-substituted lysine and homolysine analogues have identified potent inhibitors of human nitric oxide synthase enzymes, including examples with marked selectivity for the inducible isoform.

Changes in plasma carotenoid and vitamin E profile during supplementation with oil palm fruit carotenoids.

It has been hypothesized that supplementation with a single source of carotenoids causes perturbations in the plasma level of diet-derived carotenoids and that this may explain the lack of association between disease rates and the intake of carotenoid supplements. This article describes the effect of supplementation with an oil palm fruit extract, rich in beta-and alpha-carotene, on the plasma carotenoid profile of 15 healthy women volunteers. Volunteers were supplemented for 35 days with 15 mg/d of total carotenoids. Blood samples were taken at regular intervals during the supplementation period and analyzed for a range of carotenoids. Results indicate that the hydrocarbon carotenoid components of the supplement are absorbed and appear in the plasma disproportionately to the ratios in the supplement and that the plasma concentration of diet-derived lutein, a dihydroxy carotenoid ((3R,3S,6R)-beta,epsilon-carotene-3,3-diol), is depressed, whereas that of lycopene is unaffected. Plasma alpha-tocopherol concentrations were unaffected by supplementation. It is concluded that supplementation with carotenoids from a single source results in plasma carotenoid profile changes that are not predictable from a knowledge of supplement composition and that such changes should be monitored and considered when drawing conclusions as to the effect of carotenoid supplementation on health outcomes.

Absorption of all-trans and 9-cis beta-carotene in human ileostomy volunteers.

1. Mass balance studies were carried out in fasted ileostomy subjects (n = 5) given an oral physiological dose (10 mg) of beta-carotene [all-trans: 9-cis, 84:16 (w/w)] dispersed in vegetable oil. Blood and ileal effluent samples were collected and analysed for beta-carotene. 2. Results showed that 90% (range 97.0-74.3%) of the total beta-carotene was absorbed without measurable perturbation of plasma total beta-carotene concentration, or change in the all-trans: 9-cis beta-carotene ratio. Peak loss of beta-carotene in ileal effluent occurred at 4.9 h (range 2.9-8.4 h) postingestion, and no further loss was detected after 5.4-12.4 h, depending upon the individual. Comparison of the ratio of all trans-beta-carotene to 9 cis-beta-carotene in the test meal and effluent indicated that isomerization did not occur during passage through the gastrointestinal tract and that both isomers were similarly absorbed. However, the all-trans: 9-cis beta-carotene ratio of the plasma did not change. Reasoned assumptions allowed the construction of a mathematical model of plasma beta-carotene disposal. 3. It is concluded that physiological doses of isolated all-trans and 9-cis beta-carotene are well absorbed without necessarily causing detectable excursions in plasma beta-carotene concentrations, or altering the ratio of all-trans to 9-cis beta-carotene. Isomerization of beta-carotene does not occur during passage through the gastrointestinal tract. Absorbed beta-carotene is rapidly cleared from the plasma to an unobservable pool at a rate similar to that of chylomicron triacylglycerol.

The correlation between the intake of lutein, lycopene and beta-carotene from vegetables and fruits, and blood plasma concentrations in a group of women aged 50-65 years in the UK.

The correlations between the mean of 4 d weighed intakes of lutein, lycopene and beta-carotene and mean plasma concentrations during each of the four seasons were lutein r 0.64, lycopene r 0.47 and beta-carotene r 0.45. Intake was not significantly correlated with plasma concentrations during every seasonal time-point. There was a significantly higher intake of lutein during the spring compared with summer and autumn, lycopene intake was significantly higher during the summer and autumn and there were no significant seasonal differences in beta-carotene intake. There were, however, significant seasonal differences in plasma carotenoid concentrations, the highest levels occurring between May and October. There were large inter- and intra-individual variations in intake and plasma concentrations of carotenoids. BMI was inversely correlated with plasma beta-carotene (r-0.41). The findings suggest that plasma carotenoid concentrations are indicative of dietary intake, but the large intra-individual variation in plasma concentrations indicates that any assessment of longer-term status from data at any one time-point should be treated with caution.

Gene family encoding basic pathogenesis-related 1 proteins in barley.

A genomic (prb1) and two cDNA clones (PRb1-2 and PRb1-3) corresponding to two new barley basic PR-1 proteins (prb1-2 and prb1-3) were isolated from Hordeum vulgare. Genomic analysis of DNA suggests that the barley genome contains at least 6 members corresponding to the gene family encoding PR-1 proteins. Expression of these genes was induced in primary leaf tissues of the H. vulgare cv. Psaknon 4* (F14) Man. carrying Mlp resistance gene (cv. Mlp) and the near-isogenic susceptible cultivar (cv. mlp) after inoculation with Erysiphe graminis f. sp. hordei.

Pregnancy enhances cocaine's actions on the heart and within the peripheral circulation.

OBJECTIVE: Our purpose was to determine whether cocaine's enhanced cardiovascular actions in pregnancy are cardiac alone or involve the peripheral vascular system. STUDY DESIGN: Six pregnant and five nonpregnant ewes chronically instrumented for heart rate, blood pressure, cardiac output, and systemic vascular resistance were given cocaine at 1.0 and 2.0 mg/kg and monitored for 60 minutes. Blood samples for cocaine levels were taken at 5, 15, 30, and 60 minutes. RESULTS: Cocaine initially (first 60 seconds) produced increased heart rate, decreased cardiac output, decreased stroke volume, and increased cardiac oxygen consumption, which were greater in pregnant than nonpregnant ewes. After 1 minute recovery of cardiac responses was accompanied by increased systemic vascular resistance, which was greater at each dose in pregnant than nonpregnant ewes. Cocaine levels at 5 minutes for pregnant ewes were eightfold to tenfold higher than for nonpregnant ewes. CONCLUSION: Cocaine produces cardiovascular alterations that are dose and time related but, in each case, enhanced in pregnant ewes. Cocaine metabolism may contribute to this pregnancy-related phenomenon.

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts.

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 µg/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 µg/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.

Cell aggregates in wheat suspension cultures and their effects on isolation and culture of protoplasts.

Fine embryogenic suspension cultures of wheat (Triticum aestivum cv Hartog and Timmo, and T. durum cv D6962) tend to grow into large cell clumps (1-3 mm), resulting in the formation of mixed suspension cultures consisting of both fine and large cell clumps. The cell clumps were separated according to their sizes and cultured as new lines to investigate their growth rate and differentiation potential and the effects of cell aggregate size on protoplast culture. The results showed that the fine clusters (<310 µm) had a higher growth rate but a lower differentiation frequency than the large cell aggregates (310-2000 µm). After 2-4 weeks incubation, all the new lines reformed mixed suspension cultures again. The large clumps (>1100 µm) released fine cell clusters into the medium so it was possible to initiate fine embryogenic suspension cultures from the large clumps. With regard to the isolation and culture of protoplasts, although the highest yield of protoplasts was obtained from the fine cell clusters, the protoplasts isolated from different sized cell aggregates all had similar potential for sustained cell division and plant regeneration.