Search

Host cells respond to infection by generating nitric oxide (NO) as a cytotoxic weapon to facilitate killing of invading microbes. Bacterial flavohaemoglobins are well-known scavengers of NO and play a crucial role in protecting animal pathogens from nitrosative stress during infection. Erwinia chrysanthemi, which causes macerating diseases in a wide variety of plants, possesses a flavohaemoglobin (HmpX) whose function in plant pathogens has remained unclear. Here we show that HmpX consumes NO and prevents inhibition by NO of cell respiration, indicating a role in protection from nitrosative stress. Furthermore, infection of Saintpaulia ionantha plants with an HmpX-deficient mutant of E. chrysanthemi revealed that the lack of NO scavenging activity causes the accumulation of unusually high levels of NO in host tissue and triggers hypersensitive cell death. Introduction of the wild-type hmpX gene in an incompatible strain of Pseudomonas syringae had a dramatic effect on the hypersensitive cell death in soya bean cell suspensions, and markedly reduced the development of macroscopic symptoms in Arabidopsis thaliana plants. These observations indicate that HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death programme.

Ten 7-8-year-old children with specific language impairment participated in a 6-week program of narrative-based language intervention (NBLI) in an effort to evaluate NBLI's feasibility. Each intervention session targeted story content as well as story and sentence form using story retell and generation tasks. Eight children achieved the clinically significant improvement criterion from pre- and posttest comparisons of at least 1.45 points on a narrative quality (NQ) rating (p<.013). Throughout the NBLI program, the children were informally observed to show increased self-confidence in their narrative production skills. Nearly all children preferred story generation activities over story retell tasks, while story retell tasks were favored over sentence imitation drills. Pre- and posttest comparisons for number of different words, developmental sentence score, and a sentence imitation task were nonsignificant. This indicated no further evidence of positive outcomes for NBLI. Based on the significant findings for NQ, NBLI is worthy of further investigation. Modifications to enhance its ability to produce positive gains are discussed.

The 1918 influenza pandemic killed 20-40 million people worldwide, and is seen as a worst-case scenario for pandemic planning. Like other pandemic influenza strains, the 1918 A/H1N1 strain spread extremely rapidly. A measure of transmissibility and of the stringency of control measures required to stop an epidemic is the reproductive number, which is the number of secondary cases produced by each primary case. Here we obtained an estimate of the reproductive number for 1918 influenza by fitting a deterministic SEIR (susceptible-exposed-infectious-recovered) model to pneumonia and influenza death epidemic curves from 45 US cities: the median value is less than three. The estimated proportion of the population with A/H1N1 immunity before September 1918 implies a median basic reproductive number of less than four. These results strongly suggest that the reproductive number for 1918 pandemic influenza is not large relative to many other infectious diseases. In theory, a similar novel influenza subtype could be controlled. But because influenza is frequently transmitted before a specific diagnosis is possible and there is a dearth of global antiviral and vaccine stores, aggressive transmission reducing measures will probably be required.

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H(2)O(2) probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K(m) for oxygen was 80 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O(2). Purified Hmp consumed NO in the absence of added FAD (approx. 1 O(2) per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O(2) consumption required approx. 47 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx. 30 microM O(2)). On exhaustion of O(2), NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.

Respiration of Escherichia coli catalyzed either by cytochrome bo' or bd is sensitive to micromolar extracellular NO; extensive, transient inhibition of respiration increases as dissolved oxygen tension in the medium decreases. At low oxygen concentrations (25-33 microm), the duration of inhibition of respiration by 9 microm NO is increased by mutation of either oxidase. Respiration of an hmp mutant defective in flavohemoglobin (Hmp) synthesis is extremely NO-sensitive (I(50) about 0.8 microm); conversely, cells pre-grown with sodium nitroprusside or overexpressing plasmid-borne hmp(+) are insensitive to 60 microm NO and have elevated levels of immunologically detectable Hmp. Purified Hmp consumes O(2) at a rate that is instantaneously and extensively (>10-fold) stimulated by NO due to NO oxygenase activity but, in the absence of NO, Hmp does not contribute measurably to cell oxygen consumption. Cyanide binds to Hmp (K(d) 3 microm). Concentrations of KCN (100 microm) that do not significantly inhibit cell respiration markedly suppress the protection of respiration from NO afforded by Hmp and abolish NO oxygenase activity of purified Hmp. The results demonstrate the role of Hmp in protecting respiration from NO stress and are discussed in relation to the energy metabolism of E. coli in natural O(2)-depleted environments.

Euplokamis dunlapae responds to anterior stimulation by reversing the beat direction of its comb plate cilia and swimming rapidly backwards. It responds to posterior stimulation by swimming forwards at an accelerated rate. Video playback and laser monitoring were used to analyze changes in the pattern of ciliary beating, while electrical activity was recorded extracellularly. Escape responses occur with latencies of less than 150 ms and involve greatly increased ciliary beat frequencies. Giant axons run longitudinally along each of the eight comb rows, as shown by optical and electron microscopy. They form chains of overlapping neurons, with diameters of about 12 μm in life, and conducting at over 50 cm · s-1 as recorded with an extracellular electrode placed directly over the chain. The giant neurons are synaptically linked with smaller neurites of the general ectodermal nerve plexus, with each other, and with the ciliated cells of the comb plates. They appear to constitute a single system mediating rapid conduction of signals in either direction, but a full analysis was not attempted for lack of sufficient material. Electro-physiological examination of two other ctenophores (Pleurobrachia and Beroe) gives no indication of rapid conduction pathways, and these forms probably lack giant axons.

Cnidocysts have been examined from the tentacles of the ctenophore Haeckelia rubra (Euchlora rubra) and five species of hydrozoan narcomedusae (Solmundella bitentaculata, Aegina citrea, Solmissus marshalli, Solmissus albescens, and Cunina sp.) using TEM, both in sections and by firing whole cnidocysts onto EM grids. The study revealed that these apotrichous isorhiza cnidocysts have a novel morphology in which the intracapsular inverted tubule has five circumferential pleats when viewed in transverse section, rather than the usual three pleats. Accordingly, the definition of helicoptychoneme cnidocysts has been broadened to include both the usual three-pleated cnidocysts and these new five-pleated cnidocysts. In general, apotrichous isorhizas have subspherical capsules with a thick, bilayered wall, whose interior is nearly filled with the regularly coiled, helically folded, five-pleated inverted tubule. Upon discharge, the everted tubule is several mm long and the five circumferential pleats become manifested as five helical rows of spines running up the tubule, which has three morphologically different segments. The very short basal segment is devoid of ornamentation; the remaining proximal portion is characterized by five spirals of uniform, closely packed short spines; the long distal portion is characterized by a single spiral of regularly spaced large spines that derive from all five spirals-the five spirals are otherwise demarcated in the distal portion by 'scales' that are visible only with the electron microscope.