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.

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.

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.

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.

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.

Auditory-visual conflicts in the perceived duration of lights, tones and gaps.

The perceived duration of a short tone (1,000 or 1,500 msec) was longer than that of a separately presented light of equal length. Thus, when light and tone were presented simultaneously, there was a conflict in perceived duration. In that case, the perceived duration of an interval filled with both light and tone was close to that of an interval filled with tone alone. A silent gap in otherwise continuous tone was perceived as longer than a gap in otherwise continuous light, and the perceived duration of a gap occurring simultaneously in both light and tone was close to that of a gap in tone alone. Thus, auditory dominance occurred under the preceding conditions-that is, auditory-visual conflicts in perceived duration, whether occurring between filled intervals or gaps, were resolved in favor of the auditory modality. Visual dominance occurred only under one condition, in which the intensity of tone was reduced, and in which the perceived duration of a 500-msec light was longer than that of a 500-msec tone. The finding of auditory dominance in the perception of time runs counter to the results of studies of sensory conflicts in spatial perception, where vision typically dominates audition and touch.

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.

Pasture levels of tryptophan in relation to outbreaks of fog fever.

Oral administration of tryptophan induces in cattle clinical signs and lung lesions similar to those of fog fever. Tryptophan assays were performed by a microbiological method on various herbage samples. There was no significant difference in tryptophan content between herbage from two normal pastures and from three pastures in which outbreaks of fog fever occurred.

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.

Metabolic regulation in diseased leaves. I. The respiratory rise in barley leaves infected with powdery mildew.

Photosynthetic and respiratory activities have been measured in leaves of Hordeum vulgare L. var. Manchuria (barley) after infection with Erysiphe graminis var. hordei (powdery mildew). Two isogenic lines, one resistant to infection and the other highly susceptible, were examined.These isogenic lines showed very different physiological responses following infection. Photosynthesis and the chlorophyll content of resistant leaves was unaffected by infection. Respiration increased slightly and this was accompanied by small increases in activities of enzymes of glycolysis, the pentose-P pathway and the tricarboxylic acid cycle.The infection of susceptible leaves resulted in a slight increase in photosynthesis 48 hours after inoculation, but subsequently there was a progressive decrease in the photosynthesis of these leaves compared with that of noninfected leaves. The capacity of infected leaves for partial reactions of photosynthesis such as the Hill reaction and the photoreduction of nicotinamide adenine dinucleotide phosphate (NADP(1)) decreased during the later stages of infection. The levels of chlorophyll, NADPH-diaphorase and aldolase also declined. There was no detectable difference in the respiration of infected and noninfected leaves until 48 hours after inoculation. After this time, the infected leaves showed a higher respiration, the maximum difference occurring about 144 hours after inoculation. The respiratory increase was not accompanied by significant changes in the levels of enzymes of glycolysis and the tricarboxylic acid cycle with the exception of malate dehydrogenase which was lower in infected leaves. In contrast, the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase showed changes similar to that observed for respiration.The respiration and the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase did not increase in infected leaves of etiolated plants, even when excellent growth of the fungus was established by growing the plants in White's basal medium supplemented with sucrose. The respiration of a susceptible mutant barley (the yellow-green virescent mutant of the variety Himalaya) when grown in the light at 11 degrees was not changed by infection although the characteristic respiratory rise occurred in plants grown at 15 degrees . At the lower temperature chloroplasts fail to develop in this mutant, although development is normal at 15 degrees .It is suggested that the pathogen is not directly responsible for the increase in respiration in green leaves, rather that this is a response in the host cells to a loss of photosynthetic capacity.

Metabolic Regulation in Diseased Leaves II. Changes in Nicotinamide Nucleotide Coenzymes in Barley Leaves Infected With Powdery Mildew.

Nicotinamide nucleotide coenzymes were estimated spectrophotometrically in noninfected barley leaves and leaves infected with Erysiphe graminis var hordei (powdery mildew). Amounts of NADH, NADP(+) and NADPH were not altered by infection. In contrast, the NAD(+) content rose sharply and at 144 hours was 100% greater than in noninfected leaves. The respiratory rate was increased in infected leaves and the pattern of this increase was similar to that of NAD(+).The effect of infection on the intracellular distribution of NADP(+) was examined by fractionating lyophilized leaves in a nonaqueous medium. In noninfected leaves almost all of the NADP(+) was localized in the chloroplasts. In infected leaves where some chloroplast breakdown occurs, about 60% of the NADP(+) was detected in the nonchloroplast part of the cell. This intracellular redistribution of NADP(+) is discussed in relation to the increased pentose-P pathway activity occuring after infection.

Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide-adenine dinucleotide in barley.

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.

Identification of glucitol (sorbitol) and ribitol in a rust fungus, Puccinia graminis f. sp. tritici.

An examination of the soluble carbohydrates of the wheat stem rust fungus, Puccinia graminis Pers. f. sp. tritici Erikss. & E. Henn., showed the presence of glucitol (sorbitol), ribitol, fructose, and traces of xylitol, as well as confirming the presence of minnitol, arabitol, trehalose, inositol and erythritol. Ribitol and glucitol were major components in glucose-grown mycelium, and appeared to be the major components in mycelium parasitic on wheat leaves, but not in germinated or ungerminated uredospores. It is suggested that glucitol and ribitol may be intermediates (or by-products) of glucose utilization, whereas mannitol, arabitol and trehalose represent storage carbohydrates.

Nutrient content of milk and milk products: water soluble vitamins in baby milk formulae.

A study of the content of water soluble vitamins in the 12 baby milk formulae available in the UK in April 1983 showed that levels of vitamins in the individual brands were generally in excess of those declared by the manufacturer. For cows' milk based formulae the mean excess of vitamins over the declared level was 94%. Vitamin levels in prepared formulae averaged three times the minimum recommended level. The average level of B vitamins in prepared cows' milk based formulae was six times higher than in mature human milk. For the two soya based products, the levels of vitamins were generally in excess of the declared level and levels of vitamin B12, biotin and, in one sample, vitamin B6 were considerably in excess of those found in mature human milk.

A cDNA clone for a pathogenesis-related protein 1 from barley.

A barley cDNA clone (PRb-1) corresponding to an mRNA differentially induced in resistant compared to susceptible barley cultivars by powdery mildew infection was isolated and characterised. The deduced amino acid sequence revealed 24 amino acids comprising the signal peptide and 140 amino acids of the mature peptide (15 kDa). This showed close homology to PR-1-like proteins, which have been isolated from maize, tobacco, tomato and Arabidopsis thaliana. Northern blot analysis showed accumulation of the corresponding mRNA 12 h after inoculation of resistant barley cultivars with Erysiphe graminis. Increased expression of the PRb-1 gene was also observed in resistant compared with near-isogenic susceptible barley plants following treatment with ethylene, salicylic acid, methyl jasmonate and 2,6-dichloro-isonicotinic acid.

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.