bluuebunny/crossref_metadata_2025_split huggingface dataset:
Site‐specific N‐linked glycosylation analysis on the human salivary mucin MUC5B using Precursor Ion Discovery on the CAPLC Q‐TOF system
Authors: Ritchie Mark , Knight David , Kirkham Sara , Thornton David 
Published: August 2004

Introduction  Mucins are large highly O‐glycosylated oligomeric glycoproteins that form the polymer matrix of mucus, which forms part of the innate immune system. MUC5B, the predominant gel‐forming mucin in saliva, is around 600 kDa in mass before glycosylation. O‐linked glycosylation is abundant in defined repeated domains and may account for 80% of the observed mass of the secreted. Not much is known about the N‐linked glycosylation, although there are at least 34 potential sites. Despite little being known about the structure, N‐glycosylation has been shown to be important but not essential for mucin synthesis (Perez‐Vilar & Hill 1999). Here, we aim to study the N‐linked glycosylation site specifically using mass spectrometryMaterials and methods  A sample of purified human MUC5B mucin was digested with trypsin and separated by size exclusion chromatography (Thornton et al. 1999). The previously uncharacterized fractions were analysed by online LC Q‐Tof MS in order to characterize any N‐glycosylated peptides present. Glycopeptide discovery was steered by the Precursor Ion Discovery function, which detects potential glycopeptides by looking for sugar oxonium ions during intermittent high‐collision energy surveys. Candidate ions are then selected for MSMS, which reveals both the carbohydrate content and the glycosylation site location.Results  Glycopeptides were located and site specifically analysed. Data suggests N‐linked occupancy of numerous sites, and microheterogeneity was observed.Discussion  This is the first study to investigate the N‐glycans on oligomeric gel‐forming mucins. The N‐glycans are localized in the N‐ and C‐terminal portions of the MUC5B polypeptide in areas which have sparse O‐glycosylation. These regions of the molecule are known to be involved in the disulfide bond‐mediated oligomerization of the mucin monomers. Furthermore, they may be potential sites for interaction with other components of saliva that are important for biological function. It may be that they have a role in stabilizing these important regions of the molecule, or they may be sites of interaction with other salivary components.
Investigating the role of heparin sulfate proteoglycans in hereditary multiple exostoses (HME) tumourigenesis
Authors: Scholefield Z.M. , Hassell J.r , West L. , Govindraj P. , Hecht J.T. , Rushton G. , Oldershaw R. , Hardingham T. , Merry C.L.R. , Gallagher J.T. 
Published: August 2004

Introduction  Heparin sulfate (HS) has long been implicated in the bone deformity hereditary multiple exostoses (HME), and it is now clear that HME is associated with mutations in the HS biosynthetic genes EXT1 and EXT2. Interestingly, HME is also associated with an increased risk of chondro‐ and osteo‐sarcomas.Methods and results  Preliminary analysis of GAG samples purified from fibroblasts of both HME and isolated non‐HME exostoses patients reveal a dramatic shift in the ratio of CS : HS, with the HME and isolated cases having a much higher proportion of CS relative to normal controls. This is true in the case of both shed and cell surface material but is far more extreme in the latter, with the HS reducing from approximately 45% in the controls to less than 10% in HME patients. Initial analysis also reveals shortened chain length within these samples; indeed they often have two populations of chains present. Simple analysis of the total disaccharide composition of these samples demonstrates no significant differences against controls. However, detailed analysis of the subpopulations of chains (as determined by chain length) within these samples as well as cartilaginous samples from exostoses patients may provide further insight into the changes that occur within the biosynthetic pathway following disrupted EXT function.We are also carrying out immunocytochemistry with a variety of HS‐specific antibodies with the aim to further investigate normal HS structure and localization. This is being carried out on human primary chondrocytes isolated from normal patients and also adult mesenchymal stem cells as they undergo differentiation into chondrocytes. HS has been identified in both these cell types, and it is hoped that the manipulation of these cells through RNAi of different enzymes of the HS biosynthetic pathway will provide a suitable model for studying what changes may occur in cellular HS structures over the initial differentiation process in the growth plate.Discussion  Together, these investigations should provide a good model to allow us to determine the role of HS in chondrocyte differentiation and maturation in both normal and diseased states.
Epitope mapping of proteins for heparin binding sites
Authors: Vivès Romain R. , Crublet Elodie , Andrieu Jean‐Pierre , Gagnon Jean , Lortat‐Jacob Hugues 
Published: August 2004

Introduction  Heparan sulfate (HS) is a cell surface and basement membrane, sulfated polysaccharide involved in a huge array of biological processes, such as cell proliferation, chemo‐attraction, inflammation, matrix assembly, embryo development or viral attachment. These multiple functions stem from HS ability to bind and modulate the activity of a large number of proteins. The interaction of HS with its ligands is primarily, although not exclusively, of an electrostatic nature and involves the recognition of basic domains on the protein by specific motifs of the polysaccharide. Importantly, subtle differences in HS structure have been shown to dramatically affect the polysaccharide activity, as observed for HS regulation of bFGF signalling. Therefore, the identification of both protein and saccharide structural determinants implicated in HS/protein binding is critical for understanding the biological role of the interaction. In this study, we have developed a simple technique enabling the identification of HS‐binding sites on proteins.Materials and methods  Briefly, proteins were immobilized on heparin beads, using an adapted protocol of a zero‐length two‐step cross‐linking method and submitted to proteolytic digestion. Peptides retained on beads, i.e. containing amino acids involved in heparin binding, were then identified by solid phase N‐terminus protein sequencing performed directly on the heparin beads.Results  This technique was used to analyse a number of HS protein ligands, including the envelope protein gC of a herpes virus (pseudorabies) and the chemokine RANTES. Results obtained pinpointed residues known to be critical for HS binding and thus validate the method.Conclusion  This technique constitutes a novel tool that should greatly facilitate HS binding site cartography of the ever‐expanding family of heparin‐binding proteins and provides an attractive alternative to more complex approaches such as site‐directed mutagenesis.
Alveolar and plasma concentrations of interleukin‐8 and vascular endothelial growth factor following oesophagectomy
Authors: Cree R. T. J. , Warnell I. , Staunton M. , Shaw I. , Bullock R. , Griffin S. M. , Baudouin S. V. 
Published: August 2004

SummaryThe acute respiratory distress syndrome occurs in approximately 10% of all patients undergoing elective oesophagectomy. Local increases in lung pro‐inflammatory cytokines have been previously detected in high‐risk patients before the development of the acute respiratory distress syndrome. We hypothesised that similar changes would occur following oesophagectomy. Two groups of patients were studied. In the collapsed lung group (n = 11), interelukin‐8 and vascular endothelial growth factor were measured in bronchoalveolar lavage samples obtained from the intra‐operative collapsed lung after operation. In the ventilated lung group (n = 10), bronchoalveolar lavage was performed after operation from the ventilated lung and cytokines measured. Cytokines were also measured in peripheral blood samples before and after operation. Bronchoalveolar lavage cytokine levels in both lungs were of an order of magnitude greater than in peripheral blood. Pulmonary pro‐inflammatory cytokine release occurs following oesphageal surgery and may indicate subclinical lung injury.
Interobserver reliability between a nurse and anaesthetist of tests used for predicting difficult tracheal intubation
Authors: Hilditch W. G. , Kopka A. , Crawford J. M. , Asbury A. J. 
Published: August 2004

SummaryWe examined the interobserver reliability, between a nurse and anaesthetist, of five tests used to predict difficult tracheal intubation: mouth opening; thyromental distance; head and neck movement; mandibular luxation; and assessment of oropharyngeal view. For each test, an anaesthetic nurse and a specialist registrar anaesthetist were trained to use a standard method of examination. Most of the tests had either good or very good reliability. Assessment of mouth opening demonstrated only moderate reliability and assessment of oropharyngeal view demonstrated poor reliability. The interobserver reliability estimates between a nurse and an anaesthetist are similar to those previously demonstrated between two anaesthetists.
Epidural clonidine, bupivacaine and methadone as the sole analgesic agent after thoracotomy for lung resection*
Authors: Matot Idit , Drenger Benjamin , Weissman Charles , Shauli Aharona , Gozal Yaacov 
Published: August 2004

SummaryThoracic epidural analgesia can effectively relieve post‐thoracotomy pain but may also adversely affect pulmonary function. This randomised, prospective study compared the effects on pulmonary function of three different epidural analgesics (clonidine, bupivacaine and methadone). Forty‐seven patients undergoing thoracotomy were treated postoperatively for 72 h with one of the study drugs. Doses were titrated to maintain visual analogue pain scale values below 4 out of 10. Throughout the postoperative period, reductions of up to 70% of the pre‐operative value were observed in forced expiratory volume in 1 s, forced vital capacity and peak expiratory flow rate. Patients who received clonidine showed significantly faster recovery rates of forced expiratory variables compared to other patients, and by the third postoperative day significantly higher spirometry values (10–15%) were recorded in this group. As clonidine was the most effective drug in terms of preservation of pre‐operative lung function, it may be clinically advantageous in post‐thoracotomy patients.
Left lateral vs. supine, wedged position for development of block after combined spinal‐epidural anaesthesia for Caesarean section*
Authors: Lewis N. L. , Ritchie E. L. , Downer J. P. , Nel M. R. 
Published: August 2004

SummaryThis prospective, randomised controlled trial compared a spinal block developing in the left lateral position (LL), after siting a combined spinal‐epidural for Caesarean section with the woman sitting – using intrathecal hyperbaric bupivacaine and fentanyl – with a block developing in the supine, wedged position (SW). The median (IQR) block onset times for loss of cold sensation to T4 bilaterally for LL and SW women were 15 (13–19) and 12 (10.8–14) min, respectively (p = 0.004). Unilateral blocks did not occur in the left lateral group. There were no other significant differences between groups, except the median [range] 1‐min Apgar scores, which were 9 [8–10] and 9 [6–9] in the LL and SW groups, respectively (p = 0.04). In summary, the LL position is associated with a relatively slower block onset, but produces a spinal block with similar characteristics to that obtained in the SW position.
Accidental tenfold overdose of propofol in a 6‐month old infant undergoing elective craniosynostosis repair*
Authors: Patermann B. , Buzello S. , Dück M. , Paul M. , Kampe S. 
Published: August 2004

SummaryWe report a 6‐month‐old male infant undergoing elective craniosynostosis repair who accidentally received a tenfold dose of propofol over a 4‐h operative period. Myocardial dysfunction was observed after nearly 3 h of infusion; this could not solely be explained by the propofol overdose.
A randomised controlled trial of paediatric conscious sedation for dental treatment using intravenous midazolam combined with inhaled nitrous oxide or nitrous oxide/sevoflurane
Authors: Averley P. A. , Girdler N. M. , Bond S. , Steen N. , Steele J. 
Published: August 2004

SummaryFailure of dental treatment due to anxiety is a common problem in children. The aim of this study was to establish whether the use of a combination of intravenous midazolam with inhalation agents (nitrous oxide alone or in combination with sevoflurane) was any more likely to result in successful completion of treatment than midazolam alone. A further aim was to evaluate the clinical viability of these techniques as an alternative to general anaesthesia. In total, 697 children too anxious for management with relative analgesia and requiring invasive dental procedure for which a general anaesthetic would usually be required, were recruited and randomly assigned to one of three groups given the following interventions: group 1 – a combination of inhaled medical air and titrated intravenous midazolam, group 2 – a combination of inhaled 40% nitrous oxide in oxygen and titrated intravenous midazolam, and group 3 – a combination of an inhaled mixture of sevoflurane 0.3% and nitrous oxide 40% in oxygen with titrated intravenous midazolam. The primary outcome measure was successful completion of the intended dental treatment with a co‐operative child responsive to verbal commands. In group 1, 54% (94/174 children) successfully completed treatment. In group 2, 80% (204/256 children) and in group 3, 93% (249/267 children) completed treatment. This difference was significant at the 1% level. Intravenous midazolam, especially in combination with inhaled nitrous oxide or sevoflurane and nitrous oxide, are effective techniques, with the combination of midazolam and sevoflurane the one most likely to result in successful treatment.
Expression, by functional proteomics, of spontaneous tolerance in rat orthotopic liver transplantation
Authors: Pan Tai‐Long , Wang Pei‐Wen , Huang Chao‐Chen , Goto Shigeru , Chen Chao‐Long 
Published: August 2004

SummaryOrthotopic liver transplants (OLT) performed in certain combinations of donor and recipient rat strains, such as DA (RT1a) to PVG (RT1c), without immunosuppressive drugs could completely overcome major histocompatibility complex barriers. Although other organs transplanted in a similar fashion within the same combination have been promptly rejected, 60 day post‐OLT serum (POD 60) has been proven competent in rapidly reversing the established rejection in animal models. In order to understand the functional role of tolerogenic serum proteins and their involvement with immune response regulation, a comprehensive analysis surveying global changes in complex OLT systems by proteomic techniques was applied. The results display the varying protein expressions in sera extracted from naïve and transplanted animals on POD 60 with regard to immunosuppression. Among these proteins, haptoglobin (Hp) which is related to inhibition of T‐cell proliferation was found to be up‐regulated following OLT. In addition, the transcriptional expression level and intracellular localization of Hp correlated with the immune events. Hp also exhibited a strong in vitro immunosuppressive effect on the mixed lymphocyte reaction. In conclusion, the presence of Hp may play an important role in modulating the spontaneous tolerance of liver transplantation. Furthermore, the serum proteome map could provide guidance with respect to discovering potential protein targets in OLT tolerance and eventually prolong hepatic allograft survival in the future.
Allergen‐independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis
Authors: Rhee Chae‐Seo , Libet Lev , Chisholm Dugald , Takabayashi Kenji , Baird Stephen , Bigby Timothy D. , Lee Chul Hee , Horner Anthony A. , Raz Eyal 
Published: August 2004

SummaryWhile effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS‐ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty‐four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS‐ODN or control oligodeoxynucleotide (C‐ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS‐ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS‐ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C‐ODN. In addition, splenocytes from ISS‐ODN but not C‐ODN treated mice displayed attenuated OVA‐specific interleukin (IL)‐4, IL‐5, and IL‐13 but increased interferon‐γ responses. Finally, ISS‐ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2‐biased immune parameters towards Th1 dominance. As ISS‐ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS‐ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.
Immune regulatory effect of pHSP65 DNA therapy in pulmonary tuberculosis: activation of CD8+ cells, interferon‐γ recovery and reduction of lung injury
Authors: Bonato V. L. D. , Gonçalves E. D. C. , Soares E. G. , Júnior R. R. Santos , Sartori A. , Coelho‐Castelo A. A. M. , Silva C. L. 
Published: August 2004

SummaryA DNA vaccine based on the heat‐shock protein 65 Mycobacterium leprae gene (pHSP65) presented a prophylactic and therapeutic effect in an experimental model of tuberculosis. In this paper, we addressed the question of which protective mechanisms are activated in Mycobacterium tuberculosis‐infected mice after immune therapy with pHSP65. We evaluated activation of the cellular immune response in the lungs of infected mice 30 days after infection (initiation of immune therapy) and in those of uninfected mice. After 70 days (end of immune therapy), the immune responses of infected untreated mice, infected pHSP65‐treated mice and infected pCDNA3‐treated mice were also evaluated. Our results show that the most significant effect of pHSP65 was the stimulation of CD8+ lung cell activation, interferon‐γ recovery and reduction of lung injury. There was also partial restoration of the production of tumour necrosis factor‐α. Treatment with pcDNA3 vector also induced an immune stimulatory effect. However, only infected pHSP65‐treated mice were able to produce significant levels of interferon‐γ and to restrict the growth of bacilli.
Enhancement of the synthetic ligand‐mediated function of liver NK1.1Ag+ T cells in mice by interleukin‐12 pretreatment
Authors: Habu Yoshiko , Uchida Takefumi , Inui Takuo , Nakashima Hiroyuki , Fukasawa Masashi , Seki Shuhji 
Published: August 2004

SummaryWe previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin‐12 (IL‐12) act as anti‐tumour/anti‐metastatic effectors. However, IL‐12 reportedly induces a rapid disappearance of liver NKT cells by activation‐induced apoptosis. In the present study, however, we show that injection of IL‐12 into mice merely down‐regulates the NK1.1 expression of liver NKT cells and Vβ8+ intermediate T‐cell receptor cells and CD1d/α‐galactosylceramide (α‐GalCer)‐tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL‐12‐pretreated (24 hr before) mice were injected with α‐GalCer, not only serum interferon‐γ but also serum IL‐4 concentrations increased several‐fold in comparison to the control α‐GalCer‐injected mice. However, IL‐12 pretreatment markedly up‐regulated serum ALT levels and Fas‐ligand expression on NKT cells after α‐GalCer injection in middle‐aged mice only. Consistently, the liver mononuclear cells (MNC) from IL‐12‐pretreated mice stimulated with α‐GalCer in vitro produced much greater amounts of interferon‐γ and IL‐4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate‐buffered saline. Liver MNC from middle‐aged mice, but not from young mice pretreated with IL‐12, also showed increased cytotoxicity following in vitroα‐GalCer stimulation against cultured hepatocytes. Furthermore, IL‐12 treatment of middle‐aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vβ8+ T cells, and in vitro experiments also revealed that IL‐12 pretreatment of liver MNC from middle‐aged mice enhanced their tumour necrosis factor‐α production after α‐GalCer stimulation. Synthetic ligand‐mediated functions of NKT cells, including IL‐4 production, are thus enhanced by IL‐12 pretreatment.
Lipid‐associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long‐terminal repeats through Toll‐like receptors
Authors: Shimizu Takashi , Kida Yutaka , Kuwano Koichi 
Published: August 2004

SummaryMycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma‐derived lipid extracts have been reported to activate nuclear factor‐κB (NF‐κB) through Toll‐like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long‐terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid‐associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune‐deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP‐1. NF‐κB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF‐κB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse‐phase high‐performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF‐κB through TLR1 and TLR2. On the other hand, the activation of NF‐κB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.