Expression of substance P and nitric oxide synthase in vagal sensory neurons innervating the mouse airways

INTRODUCTION: Airway sensory nerves have the capacity to release neuromediators such as substance P and nitric oxide to control airway functions. The aim of the present study was to investigate substance P and neuronal nitric oxide synthase (NOS-1) expression in airway-specific sensory neurons. METHODS: Airway-projecting neurons in the jugular-nodose ganglia were investigated for NOS-1 and substance P expression by neuronal tracing and double-labelling immunoreactivity. RESULTS: Of the Fast blue labelled neurons, 14.6+/-1.8% (mean+/-S.E.M.) were immunoreactive only for NOS-1, 3.0+/-0.3% for NOS-1 and substance P, 2.7+/-0.3% only for substance P, and 79.7+/-1.7% of the labelled neurons were nonimmunoreactive for substance P or NOS-1 but were partly positive for I-B4-lectin-binding. Fast blue labelled NOS and/or substance P-positive neurons were small to medium sized (<20 microm). CONCLUSION: Based on the expression of substance P and nitric oxide synthase in airway neurons, the present study suggests that there may be substance P and NO biosynthesis and release following a peripheral activation of the afferents, there could be a triggering of substance P and NO-mediated phenomena, including those related to airway inflammation, such as plasma extravasation and vasodilatation.     

Neural mechanisms of human texture processing: texture boundary detection and visual search

Texture of various appearances, geometric distortions, spatial frequency content and densities is utilized by the human visual system to segregate items from background and to enable recognition of complex geometric forms. For automatic, or pre-attentive, segmentation of a visual scene, sophisticated analysis and comparison of surface properties over wide areas of the visual field are required. We investigated the neural substrate underlying human texture processing, particularly the computational mechanisms of texture boundary detection. We present a neural network model which uses as building blocks model cortical areas that are bi-directionally linked to implement cycles of feedforward and feedback interaction for signal detection, hypothesis generation and testing within the infero-temporal pathway of form processing. In the spirit of Jake Beck's early investigations our model particularly builds upon two key hypotheses, namely that (i) texture segregation is based on boundary detection, rather than clustering homogeneous items, and (ii) texture boundaries are detected mainly on the basis of larger scenic contexts mediated by higher cortical areas, such as area V4. The latter constraint provides a basis for element grouping in accordance to the Gestalt laws of similarity and good continuation. It is shown through simulations that the model integrates a variety of psychophysical findings on texture processing and provides a link to the underlying physiology. The functional role of feedback processing is demonstrated by context dependent modulation of V1 cell activation, leading to sharply localized detection of texture boundaries. It furthermore explains why pre-attentive processing in visual search tasks can be directly linked to texture boundary processing as revealed by recent EEG studies on visual search.     

Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomolgus monkeys (Macaca fascicularis) are independent of different oil vehicles

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg body weight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg body weight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.     

Peptide-binding motif of HLA-A*6603

The peptide motif of HLA-A*6603 was determined and compared with the available data on the peptide motifs of A*6601 and A*6602. A*6601 differs from A*6602 by two amino acids at positions 90 (Asp90Ala; outer loop) and 163 (Arg163Glu; pocket A). A*6603 differs from A*6601 and A*6602 by a single amino-acid exchange at position 70 (His70Gln; pockets A, B and C). No significant differences were found between the A*6602 and A*6603 peptide motifs suggesting that the Gln70His variation is of minor importance. However, the auxiliary anchors at position P1 of peptides bound by A*6601 (polar/acidic: Asp, Glu) and A*6602/6603 (polar/neutral: Ser) had striking differences. This finding may be best explained by the Arg163Glu substitution that results in a shift towards higher acidity in pocket A of A*6602/6603, apparently leading to the loss of preference for acidic auxiliary anchors. The similarity of A*6602 and A*6603 peptide motifs suggests low allogenicity when mismatched in stem cell transplantation. Inversely, the differences in A*6601 versus A*6602/6603 peptide motifs suggest that mismatches will have a higher allogenicity. These data will contribute to both assessing permissive mismatches in the A*66 group and weighting the impact of this individual amino-acid variation for matching and peptide binding algorithms.     

13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex

Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.     

Nuclear translocation of the heterotrimeric CCAAT binding factor of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> is dependent on two redundant localising signals in a single subunit

The CCAAT-binding complex in the Aspergillus species, also known as the Hap complex, consists of at least three subunits, namely HapB, HapC and HapE. Each Hap subunit contains an evolutionary conserved core domain. Recently, we have found that the HapC and HapE subunits do not carry a nuclear localisation signal. Furthermore, when in complex with HapB, they are transported into the nucleus via a ‘piggy back mechanism’ in A. nidulans. To extend our findings to other filamentous fungi, we examined the nuclear localisation of the A. oryzae Hap subunits by analysing several GFP fusion proteins with these Hap subunits in the hap deletion strains of A. nidulans. The nuclear translocation of the A. oryzae complex was found to be dependent on two redundant localising signals in HapB.     

In vitro degradation of the antimicrobial human peptide HEM-gamma 130-146 in plasma analyzed by a validated quantitative LC-MS/MS procedure

In stability studies during preclinical drug development, the human antimicrobial peptide hHEM-gamma 130-146 shows progressive N-terminal degradation in plasma. To determine this effect, we developed and validated a selective and quantitative muHPLC-MS/MS procedure for this compound. Following deproteinization by precipitation, reversed-phase separation is performed with a time-saving two-column design online coupled to an ion trap mass spectrometer for electrospray ionization MS detection. Using a linear calibration curve obtained with synthetic external standards ranging nearly two orders of magnitude, we achieved good precision (repeatability and reproducibility: 5-15%), accuracy (-3 to 15%), and ruggedness with a lower limit of quantification at 0.29 microg/ml plasma (0.15 microM). Because of good linearity (r2>0.999), the recovery (84+/-3%) and ion suppression (86+/-4% remaining intensity) were calculated from specifically prepared calibration curves. The developed procedure was applied to human and animal plasma samples. Incubations in the presence and absence of proteinase inhibitors revealed at least an aminopeptidase M activity for the initial N-terminal truncation of tryptophan (W130) and a putative glutaminyl-peptide cyclotransferase activity for the resulting intermediate starting with the bared glutamine residue (Q131). The calculated periods of half-change demonstrated exceeding interspecies variations, whereas the intraspecies variations were only between 20 and 30%. The current procedure is valuable as a generic method for pharmaceutical purposes, and data give important information for further development toward a potential natural drug candidate.