Photoinactivation of Ammonia Oxidation in Nitrosomonas

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.     

Protective Ventilation of Preterm Lambs Exposed to Acute Chorioamnionitis Does Not Reduce Ventilation-Induced Lung or Brain Injury

Background

The onset of mechanical ventilation is a critical time for the initiation of cerebral white matter (WM) injury in preterm neonates, particularly if they are inadvertently exposed to high tidal volumes (V_T) in the delivery room. Protective ventilation strategies at birth reduce ventilation-induced lung and brain inflammation and injury, however its efficacy in a compromised newborn is not known. Chorioamnionitis is a common antecedent of preterm birth, and increases the risk and severity of WM injury. We investigated the effects of high V_T ventilation, after chorioamnionitis, on preterm lung and WM inflammation and injury, and whether a protective ventilation strategy could mitigate the response.

Methods

Pregnant ewes (n = 18) received intra-amniotic lipopolysaccharide (LPS) 2 days before delivery, instrumentation and ventilation at 127±1 days gestation. Lambs were either immediately euthanased and used as unventilated controls (LPS_UVC; n = 6), or were ventilated using an injurious high V_T strategy (LPS_INJ; n = 5) or a protective ventilation strategy (LPS_PROT; n = 7) for a total of 90 min. Mean arterial pressure, heart rate and cerebral haemodynamics and oxygenation were measured continuously. Lungs and brains underwent molecular and histological assessment of inflammation and injury.

Results

LPS_INJ lambs had poorer oxygenation than LPS_PROT lambs. Ventilation requirements and cardiopulmonary and systemic haemodynamics were not different between ventilation strategies. Compared to unventilated lambs, LPS_INJ and LPS_PROT lambs had increases in pro-inflammatory cytokine expression within the lungs and brain, and increased astrogliosis (p<0.02) and cell death (p<0.05) in the WM, which were equivalent in magnitude between groups.

Conclusions

Ventilation after acute chorioamnionitis, irrespective of strategy used, increases haemodynamic instability and lung and cerebral inflammation and injury. Mechanical ventilation is a potential contributor to WM injury in infants exposed to chorioamnionitis.     

Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox

Two decades after a worldwide vaccination campaign was used to successfully eradicate naturally occurring smallpox, the threat of bioterrorism has led to renewed vaccination programs. In addition, sporadic outbreaks of human monkeypox in Africa and a recent outbreak of human monkeypox in the U.S. have made it clear that naturally occurring zoonotic orthopoxvirus diseases remain a public health concern. Much of the threat posed by orthopoxviruses could be eliminated by vaccination; however, because the smallpox vaccine is a live orthopoxvirus vaccine (vaccinia virus) administered to the skin, the vaccine itself can pose a serious health risk. Here, we demonstrate that rhesus macaques vaccinated with a DNA vaccine consisting of four vaccinia virus genes (L1R, A27L, A33R, and B5R) were protected from severe disease after an otherwise lethal challenge with monkeypox virus. Animals vaccinated with a single gene (L1R) which encodes a target of neutralizing antibodies developed severe disease but survived. This is the first demonstration that a subunit vaccine approach to smallpox-monkeypox immunization is feasible.     

Effect of Selenium Yeast Supplementation on Naturally Acquired Parasitic Infection in Ewes

Gastrointestinal parasites cause substantial economic losses in pasture-based sheep production systems. Supranutritional organic selenium (Se) supplementation may be beneficial because it improves immune responses to pathogens. To evaluate the effect of Se-yeast supplementation on gastrointestinal parasite load, 30 ewes per treatment group were drenched weekly with no Se, 4.9 mg Se/week as Se yeast (maximum FDA-allowed concentration), or supranutritional concentrations of Se yeast (14.7 and 24.5 mg Se/week) starting early fall for 85 weeks. Fecal samples were collected at weeks 63, 66, 78, and 84 and counted for total trichostrongyle-type eggs and Haemonchus contortus eggs (in samples with ≥200 trichostrongyle eggs/g feces). During breeding season (fall), ewes were kept on pasture; ewes receiving 24.5 mg Se/week had lower fecal trichostrongyle egg counts (93?±?40 eggs/g feces) compared with ewes receiving no Se (537?±?257 eggs/g feces; P?=?0.007) or ewes receiving 4.9 mg Se/week as Se yeast (398?±?208 eggs/g feces; P?=?0.03). In winter, fecal trichostrongyle egg counts decreased, and group differences were not apparent. During lambing season (spring), ewes were kept in the barn and fecal trichostrongyle egg counts increased, although no group differences were observed. However, none of the ewes receiving supranutritional Se yeast, and with trichostrongyle egg counts ≥200 eggs/g of feces, but four of the ewes receiving lower Se dosages had H. contortus egg counts ≥1,000 eggs/g feces (P?=?0.04). Our results suggest that supranutritional Se-yeast supplementation may enhance resistance to naturally occurring H. contortus gastrointestinal parasitism in sheep.     

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium,Synechocystis PCC 6803

Despite intense research, the mechanism of Cd²⁺ toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd²⁺ uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO₂‐dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd²⁺ (50% inhibition in ～15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light‐induced P700 redox transients, O₂ polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short‐ and long‐term effects, they exhibit virtually the same Cd²⁺ concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.     

Interhemispheric switching mediates perceptual rivalry

BACKGROUND: Binocular rivalry refers to the alternating perceptual states that occur when the images seen by the two eyes are too different to be fused into a single percept. Logothetis and colleagues have challenged suggestions that this phenomenon occurs early in the visual pathway. They have shown that, in alert monkeys, neurons in the primary visual cortex continue to respond to their preferred stimulus despite the monkey reporting its absence. Moreover, they found that neural activity higher in the visual pathway is highly correlated with the monkey's reported percept. These and other findings suggest that the neural substrate of binocular rivalry must involve high levels, perhaps the same levels involved in reversible figure alternations. RESULTS: We present evidence that activation or disruption of a single hemisphere in human subjects affects the perceptual alternations of binocular rivalry. Unilateral caloric vestibular stimulation changed the ratio of time spent in each competing perceptual state. Transcranial magnetic stimulation applied to one hemisphere disrupted normal perceptual alternations when the stimulation was timed to occur at one phase of the perceptual switch, but not at the other. Furthermore, activation of a single hemisphere by caloric stimulation affected the perceptual alternations of a reversible figure, the Necker cube. CONCLUSIONS: Our findings suggest that interhemispheric switching mediates perceptual rivalry. Thus, competition for awareness in both binocular rivalry and reversible figures occurs between, rather than within, each hemisphere. This interhemispheric switch hypothesis has implications for understanding the neural mechanisms of conscious experience and also has clinical relevance as the rate of both types of perceptual rivalry is slow in bipolar disorder (manic depression).     

The prion protein and neuronal zinc homeostasis

Although the prion protein (PrP) is known to be the causative agent of the neurodegenerative transmissible spongiform encephalopathies, its normal cellular function remains elusive. Octapeptide repeats in the N terminus of PrP bind metal ions and are required for the endocytosis of PrP upon exposure of cells to copper or zinc. As the concentration of zinc in the extracellular spaces of the brain is higher than that for copper, we put forward the hypothesis that PrP is involved in neuronal zinc homeostasis; PrP might be involved in transport of zinc into the cell or might act as a zinc sensor. In prion disease, when the protein undergoes a conformational change to the infectious form, this function of PrP in zinc homeostasis might be compromised.     

An NAADP-gated two-pore channel targeted to the plasma membrane uncouples triggering from amplifying Ca2+ signals

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.     

Increases in lung expansion alter pulmonary hemodynamics in fetal sheep.

Prolonged increases in fetal lung expansion stimulate fetal lung growth and development, but the effects on pulmonary hemodynamics are unknown. Our aim was to determine the effect of increased fetal lung expansion, induced by tracheal obstruction (TO), on pulmonary blood flow (PBF) and vascular resistance (PVR). Chronically catheterized fetal sheep (n = 6) underwent TO from 120 to 127 days of gestational age (term approximately 147 days); tracheas were not obstructed in control fetuses (n = 6). PBF, PVR, and changes to the PBF waveform were determined. TO significantly increased lung wet weight compared with control (166.3 +/- 20.2 vs. 102.0 +/- 18.8 g; P < 0.05). Despite the increase in intraluminal pressure caused by TO (5.0 +/- 0.9 vs. 2.4 +/- 1.0 mmHg; P < 0.001), PBF and PVR were similar between groups after 7 days (TO 28.1 +/- 3.2 vs. control 34.1 +/- 10.0 ml.min(-1).100 g lung wt(-1)). However, TO markedly altered pulmonary hemodynamics associated with accentuated fetal breathing movements, causing a reduction rather than an increase in PBF at 7 days of TO. To account for the increase in intraluminal pressure, the pressure was equalized by draining the lungs of liquid on day 7 of TO. Pressure equalization increased PBF from 36.8 +/- 5.2 to 112.4 +/- 22.8 ml/min (P = 0.01) and markedly altered the PBF waveform. These studies provide further evidence to indicate that intraluminal pressure is an important determinant of PBF and PVR in the fetus. We suggest that the increase in PBF associated with pressure equalization following TO reflects an increase in growth of the pulmonary vascular bed, leading to an increase in its cross-sectional area.