Morphine has a dual concentration-dependent effect on K+-evoked substance P release from rat peripheral airways

Our previous investigations of possible lung mechanisms underlying the effectiveness of nebulized morphine for the relief of dyspnoea, have shown a high density of non-conventional opioid binding sites in rat airways with similar binding characteristics (opioid alkaloid-sensitive, opioid peptide-insensitive) to that of putative mu(3)-opioid receptors on immune cells. To investigate whether these lung opioid binding sites are functional receptors, this study was designed to determine (using superfusion) whether morphine modulates the K+-evoked release of the pro-inflammatory neuropeptide, substance P (SP), from rat peripheral airways. Importantly, K+-evoked SP release was Ca2+-dependent, consistent with vesicular release. Submicromolar concentrations of morphine (1 and 200 nM) inhibited K+-evoked SP release from rat peripheral airways in a naloxone (1 mu M) reversible manner. By contrast, 1 mu M morphine enhanced K+-evoked SP release and this effect was not reversed by 1 mu M naloxone. However, 100 mu M naloxone not only antagonized the facilitatory effect of 1 mu M morphine on K+-evoked SP release from rat peripheral airways but it inhibited release to a similar extent as 200 nM morphine. It is possible that these latter effects are mediated by non-conventional opioid receptors located on mast cells, activation of which causes naloxone-reversible histamine release that in turn augments the release of SP from sensory nerve terminals in the peripheral airways. Clearly, further studies are required to investigate this possibility. (C) 1997 Academic Press Limited.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were: taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

T cell-mediated and non-specific inflammatory mechanisms contribute to the skin pathology of HPV 16 E6E7 transgenic mice

One of three lines of mice transgenic for the E6 and E7 genes of human papillomavirus type 16 (HPV16) expressed from an alpha A-crystallin promoter also expresses the transgene ectopically in the skin. This line, designated alpha ACE6E7#19, develops skin disease from 3 months of age, characterised by epidermal hyperplasia and eventual skin loss. Administration of complete Freund's adjuvant (CFA) to alpha ACE6E7#19 mice, but not to nontransgenic littermate controls, induced local epidermal hyperplasia which was histologically similar to the spontaneously arising skin pathology. Local application of 2,4-dinitrochlorobenzene (DNCB) to DNCB-sensitised aACE6E7#19 mice, but not DNCB-sensitised controls, also induced hyperplasia. Treatment with cyclosporin A (CsA) or systemic depletion of CD4+ cells significantly reduced the incidence of skin disease. These data suggest that local inflammation, and cytokines produced by T helper cells, contribute to the induction of hyperplastic skin disease in alpha ACE6E7#19 mice. Spontaneous skin disease with similar histological appearance, frequency, age of onset and severity in alpha ACE6E7#19 mice was observed in scid-/- aACE6E7#19 mice, despite immune paresis. Antigen-specific immune responses and T-cell cytokines a re therefore not necessary for the induction of skin disease. We propose that epidermal hyperplasia associated with HPV16 E6 and E7 expression in skin is accelerated by local secretion of pro-inflammatory cytokines, whose production can be enhanced by activated CD4+ T cells.

Optimizing electrode sites for segmental bioimpedance measurements

Recent advances in the application of bioelectrical impedance analysis (BIA) have indicated that a more accurate approach to the estimation of total body water is to consider the impedance of the various body segments rather than simply that of the whole body. The segmental approach necessitates defining and locating the physical demarcation between both the trunk and leg and the trunk and arm. Despite the use of anatomical markers, these points of demarcation are difficult to locate with precision between subjects. There are also technical problems associated with the regional dispersion of the current distribution from one segment (cylinder) to another of different cross-sectional area. The concept of equipotentials in line with the proximal aspects of the upper land lower) limbs along the contralateral limbs was investigated and, in particular, the utility of this concept in the measurement of segmental bioimpedance. The variation of measured segmental impedance using electrode sites along these equipotentials was less than 2.0% for all of the commonly used impedance parameters. This variation is approximately equal to that expected from biological variation over the measurement time. It is recommended that the electrode sites, for the measurement of segmental bioelectrical impedance in humans, described herein are adopted in accordance with the proposals of the NM Technology Assessment Conference Statement.

Implication of the visual system in the regulation of activity cycles in the absence of solar light: 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression in the brains of demersal deep-sea gadiform fish

Relative eye size, gross brain morphology and central localization of 2-[I-125]iodomelatonin binding sites and melatonin receptor gene expression were compared in six gadiform fish living at different depths in the north-east Atlantic Ocean: Phycis blennoides (capture depth range 265-1260 m), Nezumia aequalis (445-1512 m), Coryphaenoides rupestris (706-1932 m), Trachyrincus murrayi (1010-1884 m), Coryphaenoides guentheri (1030 m) and Coryphaenoides (Nematonurus) armatus (2172-4787 m). Amongst these, the eye size range was 0.15-0.35 of head length with a value of 0.19 for C.(N.) armatus, the deepest species. Brain morphology reflected behavioural differences with well-developed olfactory regions in P.blennoides, T.murrayi and C. (N.) armatus and evidence of olfactory deficit in N. aequalis, C. rupestris and C. guentheri. All species had a clearly defined optic tectum with 2-[I-125] iodomelatonin binding and melatonin receptor gene expression localized to specific brain regions in a similar pattern to that found in shallow-water fish. Melatonin receptors were found throughout the visual structures of the brains of all species. Despite living beyond the depth of penetration of solar light these fish have retained central features associated with the coupling of cycles of growth, behaviour and reproduction to the diel light-dark cycle. How this functions in the deep sea remains enigmatic.

Conformational selection of inhibitors and substrates by proteolytic enzymes: Implications for drug design and polypeptide processing

Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/ substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D H-1 NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the H-1 NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Her chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and IID-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.

Regulation of the Hsp90-binding immunophilin, cyclophilin 40, is mediated by multiple sites for CA-binding protein (GABP)

Within steroid receptor heterocomplexes the large tetraticopeptide repeat-containing immunophilins, cyclophilin 40 (CyP40), FKBP51, and FKBP52, target a common interaction site in heat shock protein 90 (HspSO) and act coordinately with HspSO to modulate receptor activity. The reversible nature of the interaction between the immunophilins and HspSO suggests that relative cellular abundance might be a key determinant of the immunophilin component within steroid receptor complexes. To investigate CyP40 gene regulation, we have isolated a fi-kilobase (kb) 5 ' -flanking region of the human gene and demonstrated that a similar to 50 base pair (bp) sequence adjacent to the transcription start site is essential for CyP40 basal expression. Three tandemly arranged Ets sites within this critical region were identified as binding elements for the multimeric Ets-related transcription factor, GA binding protein (GABP). Functional studies of this proximal promoter sequence, in combination with mutational analysis, confirmed these sites to be crucial for basal promoter function. Furthermore, overexpression of both GABP alpha and GABP beta subunits in Cos1 cells resulted in increased endogenous CyP40 mRNA levels. Significantly, a parallel increase in FKBP52 mRNA expression was not observed, highlighting an important difference in the mode of regulation of the CyP40 and FKBP52 genes. Our results identify GABP as a key regulator of CyP40 expression. GAFF is a common target of mitogen and stress-activated pathways and may integrate these diverse extracellular signals to regulate CyP40 gene expression.

Trichomonas vaginalis is associated with pelvic inflammatory disease in women infected with human immunodeficiency virus

We assessed the association between the causative agents of vaginal discharge and pelvic inflammatory disease (PID) among women attending a rural sexually transmitted disease clinic in South Africa; the role played by coinfection with human immunodeficiency virus type 1 (HIV-1) was studied. Vaginal and cervical specimens were obtained to detect Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and bacterial vaginosis. HIV-1 infection was established by use of serum antibody tests. A total of 696 women with vaginal discharge were recruited, 119 of whom had clinical PID. Patients with trichomoniasis had a significantly higher risk of PID than did women without trichomoniasis (P = .03). PID was not associated with any of the other pathogens. When the patients were stratified according to HIV-1 status, the risk of PID in HIV-1-infected patients with T. vaginalis increased significantly (P = .002); no association was found in patients without HIV-1. T. vaginalis infection of the lower genital tract is associated with a clinical diagnosis of PID in HIV-1-infected women.

Semiochemicals and social signaling in the wild European rabbit in Australia: II. Variations in chemical composition of chin gland secretion across sampling sites

The volatile components of the chin gland secretion of the wild European rabbit, Oryctolagus cuniculus (L.), were investigated with the use of gas chromatography. Studies of the chemical nature of this secretion by previous workers demonstrated that it was important in the maintenance of social structure in this species. This study identified 34 different volatile components that consist primarily of aromatic and aliphatic hydrocarbons. Especially common are a series of alkyl-substituted benzene derivatives that provide most of the compound diversity in the secretion. Samples of chin gland secretion collected from animals at three different geographical locations, separated by more than 100 km, showed significant differences in composition. This work suggests that variation among populations needs to be considered when undertaking semiochemical research. Alternate nonparametric methods are also used for the analysis of chromatographic data.

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.