Involvement of napsin A in the C- and N-terminal processing of surfactant protein B in type-II pneumocytes of the human lung

Surfactant protein B (SP-B) is a critical component of pulmonary surfactant, and a deficiency of active SP-B results in fatal respiratory failure. SP-B is synthesized by type-II pneumocytes as a 42-kDa propeptide (proSP-B), which is posttranslationally processed to an 8-kDa surface-active protein. Napsin A is an aspartic protease expressed in type-II pneumocytes. To characterize the role of napsin A in the processing of proSP-B, we colocalized napsin A and precursors of SP-B as well as SP-B in the Golgi complex, multivesicular, composite, and lamellar bodies of type-II pneumocytes in human lungs using immunogold labeling. Furthermore, we measured aspartic protease activity in isolated lamellar bodies as well as isolated human type-II pneumocytes and studied the cleavage of proSP-B by napsin A and isolated lamellar bodies in vitro. Both, napsin A and isolated lamellar bodies cleaved proSP-B and generated three identical processing products. Processing of proSP-B by isolated lamellar bodies was completely inhibited by an aspartic protease inhibitor. Sequence analysis of proSP-B processing products revealed several cleavage sites in the N- and C-terminal propeptides as well as one in the mature peptide. Two of the four processing products generated in vitro were also detected in type-II pneumocytes. In conclusion, our results show that napsin A is involved in the N- and C-terminal processing of proSP-B in type-II pneumocytes.     

Radiosensitivity of ataxia telangiectasia and Nijmegen breakage syndrome homozygotes and heterozygotes as determined by three-color FISH chromosome painting

A three-color chromosome painting technique was used to examine the spontaneous and radiation-induced chromosomal damage in peripheral lymphocytes and lymphoblastoid cells from 11 patients with ataxia telangiectasia (AT) and from 14 individuals heterozygous for an AT allele. In addition, cells from two homozygous and six obligate heterozygous carriers of mutations in the Nijmegen breakage syndrome gene (NBS) were investigated. The data were compared to those for chromosome damage in 10 unaffected control individuals and 48 cancer patients who had not yet received therapeutic treatment. Based on the well-documented radiation sensitivity of AT and NBS patients, it was of particular interest to determine whether the FISH painting technique used in these studies allowed the reliable detection of an increased sensitivity to in vitro irradiation of cells from heterozygous carriers. Peripheral blood lymphocytes and lymphoblastoid cells from both the homozygous AT and NBS patients showed the highest cytogenetic response, whereas the cells from control individuals had a low number of chromosomal aberrations. The response of cells from heterozygous carriers was intermediate and could be clearly differentiated from those of the other groups in double-coded studies. AT and NBS heterozygosity could be distinguished from other genotypes by the total number of breakpoints per cell and also by the number of the long-lived stable aberrations in both AT and NBS. Only AT heterozygosity could be distinguished by the fraction of unstable chromosome changes. The slightly but not significantly increased radiosensitivity that was found in cancer patients was apparently due to a higher trend toward rearrangements compared to the controls. Thus the three-color painting technique presented here proved to be well suited as a supplement to conventional cytogenetic techniques for the detection of heterozygous carriers of these diseases, and may be superior method.     

The effect of a taurine-containing drink on performance in 10 endurance-athletes

Summary To determine the effect of a taurine-enriched drink Red Bull on performance, 10 endurance-athletes performed three trials. After 60 min. cycling at approximately 70% VO₂ max, the subjects pedalled to exhaustion on a cycle ergometer. During each exercise, the subjects received 500 ml of a test-drink after 30 min. submaximal cycling: Red Bull without taurine, without glucuronolacton (U1), Red Bull without taurine, without glucuronolacton, without caffeine (U2) and Red Bull original drink containing taurine, glucuronolacton and caffeine (U3).The heart rate level was significantly lower in U3 (p = 0,0031) 15 min. after application. The plasma catecholamines increased slightly from begin of exercise to 15 min. after application of the drinks in all trials but remained on a significantly lower level in U3 (epinephrine (p = 0,0011) and norepinephrine (p = 0,0003). Endurance time was significantly longer with Red Bull original in U3 (p = 0,015). The results of this study show a positive effect of a taurine-containing drink on hormonal responses which leads to a higher performance.     

Deactivation of Brain Areas During Self-Regulation of Slow Cortical Potentials in Seizure Patients

This study investigates the neurophysiological basis of EEG feedback for patients with epilepsy. Brain areas are identified that become hemodynamically deactivated when epilepsy patients, trained in EEG self-regulation, generate positive slow cortical potentials (SCPs). Five patients were trained in producing positive SCPs, using a training protocol previously established to reduce seizure frequency in patients with drug refractory epilepsy. Patients attempted to produce positive SCP shifts in a functional magnetic resonance imaging (fMRI) scanner. Two patients were able to reliably produce positive SCP shifts. When these successful regulators were prompted to produce positive SCPs, blood oxygen level-dependent (BOLD) response indicated deactivation, in comparison to a control state, around the recording electrode, frontal lobe, and thalamus. Unsuccessful regulators’ BOLD response indicated no deactivation in cortical areas proximal to the active electrode. No thalamic deactivation was found in poor regulators. Decreased seizure frequency from SCP training may be the result of positively reinforced inhibition in cortical areas proximal to active electrode placement, the frontal cortex, and the thalamus.     

Mouse Models of Aerosol-Acquired Tularemia Caused by Francisella tularensis Types A and B

After preliminary assessment of virulence in AKR/J, DBA/1, BALB/c, and C57BL/6 mice, we investigated histopathologic changes in BALB/c and C57BL/6 mice infected with type A (strain SCHU S4) or type B (strain 425) Francisella tularensis by aerosol exposure. In mice exposed to type A infection, changes in histologic presentation were not apparent until day 3 after infection, when pyogranulomatous inflammation was detected in spleens and livers of BALB/c mice, and in lungs and spleens of C57BL/6 mice. Histopathologic changes were most severe and widespread in both mouse strains on day 5 after infection and seemed to completely resolve within 22 d of challenge. BALB/c mice were more resistant than C57BL/6 mice in lethal-dose calculations, but C57BL/6 mice cleared the infection more rapidly. Mice similarly challenged with type B F. tularensis also developed histopathologic signs of infection beginning on day 3. The most severe changes were noted on day 8 and were characterized by granulomatous or pyogranulomatous infiltrations of the lungs. Unlike type A infection, lesions due to type B did not resolve over time and remained 3 wk after infection. In type B, but not type A, infection we noted extensive inflammation of the heart muscle. Although no microorganisms were found in tissues of type A survivors beyond 9 d after infection, mice surviving strain 425 infection had a low level of residual infection at 3 wk after challenge. The histopathologic presentation of tularemia caused by F. tularensis types A and B in BALB/c and C57BL/6 mice bears distinct similarities to tularemia in humans.Abbreviations: LVS, live vaccine strain; SUBLN, submandibular lymph node; MESLN, mesenteric lymph nodeTularemia is an infectious disease caused by the gram-negative bacterium Francisella tularensis. Clinical disease was first seen in Japan in 1837, when people who ate meat from rabbits developed fever, chills, and glandular tumors.²⁷ In the United States, the disease was first described in 1911 by Dr Edward Francis, who observed a plague-like illness in ground squirrels. In 1914, the first human case in the United States was described in a butcher.⁴⁸ Dr Francis, for whom the microorganism was named, characterized the clinical signs, symptoms, and the mode of transmission after infection.Tularemia is one of the most pathogenic diseases known, causing disease after inoculation or inhalation of as few as 10 microorganisms.²⁷ The microorganism is infectious by many routes, including ingestion of contaminated food or water, through microabrasions or bites from various arthropods, or inhalation of the infectious agent. The species F. tularensis includes several subspecies including tularensis, holarctica, novidia, and mediaasiatica. The type A F. tularensis subspecies tularensis is highly virulent in humans and is the dominant species in North America. Type B F. tularensis subspecies holarctica (also called palearctica) is usually found in European countries and is less virulent.⁴⁵ Humans infected with either subspecies can develop an acute febrile illness.F. tularensis is a facultative intracellular pathogen and multiplies in macrophages. Target organs are associated with the reticuloendothelial system and include lymph nodes, lungs, spleen, liver, and kidneys. In human pneumonic tularemia, infection progresses systemically from the lungs to other organs and causes pathologic changes, primarily to the liver and spleen.^9,11,19,38 Humans infected by aerosol exposure also show hemorrhagic inflammation of the airways, which can progress to bronchopneumonia.⁴³ Alveolar spaces become filled with a mononuclear cell infiltrate. Pleuritis with adhesions and effusion and hilar lymphadenopathy are commonly found.³³ The fatality rate for untreated pneumonic infections is approximately 30% to 60% for type A F. tularensis and approximately 10% for infections with type B F. tularensis.⁴² Because of its high infectivity, morbidity, and mortality, this microorganism is classified as a Category A Select Agent. F. tularensis can cause natural infections in a variety of animals, including rabbits, cats, prairie dogs, voles, raccoons, squirrels, rats, lemmings, wild mice, and other species.^{1,3,4,12,30,35,40,49} Although the source of infection can be inapparent, outdoor-housed rhesus macaques can acquire tularemia.^15,37Animal models using virulent (for humans) strains are essential for the development of efficacious vaccines and evaluation of antimicrobials against pathogenic diseases affecting humans. Due to the virulence of this microorganism, most experimental infections in rodent model systems use the Live Vaccine Strain (LVS) to minimize the risk to laboratory workers,¹⁷ although many recent studies use the more virulent type B and type A strains. LVS can be lethal to mice but is attenuated in humans. LVS is lethal in naïve mice and causes pathologic reactions similar to those seen with virulent strains in humans.⁸ However, using an LVS model of infection is not without potential problems. First, dose lethality varies widely, depending on the route of LVS infection. Naïve mice are most sensitive to intraperitoneal challenge, followed by intravenous, intranasal, aerosol, and intradermal challenge. Furthermore, the LD₅₀ for LVS administered by aerosol is greater than 10³ microorganisms, whereas the LD₅₀ for virulent F. tularensis strains is less than 10 microorganisms.⁸To better understand the pathogenesis of this important Category A pathogen, we determined the virulence and investigated the histopathogenesis of type A and type B F. tularensis in mice challenged by small-particle aerosol. We compared the histopathogenesis of infection over a period of 3 wk in BALB/c and C57BL/6 mice challenged with 1468 microorganisms or 100 microorganisms of F. tularensis SCHU S4 (type A). Type B (strain 425) infection was similarly investigated in BALB/c mice challenged with 102 microorganisms. Bacterial burdens in spleens, livers, lungs, and blood were determined.     

Specific contributions of ventromedial, anterior cingulate, and lateral prefrontal cortex for attentional selection and stimulus valuation

Attentional control ensures that neuronal processes prioritize the most relevant stimulus in a given environment. Controlling which stimulus is attended thus originates from neurons encoding the relevance of stimuli, i.e. their expected value, in hand with neurons encoding contextual information about stimulus locations, features, and rules that guide the conditional allocation of attention. Here, we examined how these distinct processes are encoded and integrated in macaque prefrontal cortex (PFC) by mapping their functional topographies at the time of attentional stimulus selection. We find confined clusters of neurons in ventromedial PFC (vmPFC) that predominantly convey stimulus valuation information during attention shifts. These valuation signals were topographically largely separated from neurons predicting the stimulus location to which attention covertly shifted, and which were evident across the complete medial-to-lateral extent of the PFC, encompassing anterior cingulate cortex (ACC), and lateral PFC (LPFC). LPFC responses showed particularly early-onset selectivity and primarily facilitated attention shifts to contralateral targets. Spatial selectivity within ACC was delayed and heterogeneous, with similar proportions of facilitated and suppressed responses during contralateral attention shifts. The integration of spatial and valuation signals about attentional target stimuli was observed in a confined cluster of neurons at the intersection of vmPFC, ACC, and LPFC. These results suggest that valuation processes reflecting stimulus-specific outcome predictions are recruited during covert attentional control. Value predictions and the spatial identification of attentional targets were conveyed by largely separate neuronal populations, but were integrated locally at the intersection of three major prefrontal areas, which may constitute a functional hub within the larger attentional control network.     

Hybrid maize breeding with doubled haploids: V. Selection strategies for testcross performance with variable sizes of crosses and S1 families

In hybrid maize (Zea mays L.) breeding, doubled haploids (DH) are increasingly replacing inbreds developed by recurrent selfing. Doubled haploids may be developed directly from S₀ plants in the parental cross or via S₁ families. In both these breeding schemes, we examined 2 two-stage selecting strategies, i.e., considering or ignoring cross and family structure while selection among and within parental crosses and S₁ families. We examined the optimum allocation of resources to maximize the selection gain ΔG and the probability P(q) of identifying the q% best genotypes. Our specific objectives were to (1) determine the optimum number and size of crosses and S₁ families, as well as the optimum number of test environments and (2) identify the superior selection strategy. Selection was based on the evaluation of testcross progenies of (1) DH lines in both stages (DHTC) and (2) S₁ families in the first stage and of DH lines within S₁ families in the second stage (S₁TC-DHTC) with uniform and variable sizes of crosses and S₁ families. We developed and employed simulation programs for selection with variable sizes of crosses and S₁ families within crosses. The breeding schemes and selection strategies showed similar relative efficiency for both optimization criteria ΔG and P (0.1%). As compared with DHTC, S₁TC-DHTC had larger ΔG and P (0.1%), but a higher standard deviation of ΔG. The superiority of S₁TC-DHTC was increased when the selection was done among all DH lines ignoring their cross and family structure and using variable sizes of crosses and S₁ families. In DHTC, the best selection strategy was to ignore cross structures and use uniform size of crosses.     

Monitoring of root growth and redox conditions in paddy soil rhizotrons by redox electrodes and image analysis

The root-zone of wetland rice was monitored in a paddy soil throughout a vegetation period with the aid of a rhizotron experiment. For this purpose (a) digital images of the root-zone were taken daily, and (b) the redox potential was measured in situ every day. The images were processed by image analysis in order to display areas of oxidation and reduction in the soil. Therefore, thresholds were set to simplify the localization and quantification of discrete areas which were colourized due to the redox potential. Both, images and measured redox potentials, provide the basis for the visualization of the root and redox dynamics in the root-zone. The anaerobic root-zone of flooded paddy soils is significantly influenced by the aerenchymal transport of oxygen to rice roots. The release of oxygen into the rhizosphere, which causes different patterns of oxidized and reduced areas in the course of the vegetation period, also affects microbial communities such as methane producing archaea or methane oxidizing bacteria. The visualization of redox dynamics may, therefore, be useful to localize potential hotspots for the microorganisms in the root-zone of paddy soils. The reduced and oxidized conditions changed spatiotemporally. Oxidized areas were mostly found in the surrounding of active roots and in a distinct layer next to the soil surface. Reduced areas shifted from beneath the oxidized surface layer into sparsely-rooted soil. The ratio of the analyzed oxidized and reduced areas was oscillating with increasing intensity throughout the monitored vegetation period.