Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n=182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

Effect of induced airway inflammation in asthma : the expression of trefoil factors, secretoglobins and genes involved in histone acetylation

Asthma is an incapacitating disease of the respiratory system, which causes extensive morbidity and mortality worldwide. Asthma affects more than 300 million people globally(Masoli et al. 2004). In Australia, it affects 10.2% of the population (Masoli et al. 2004) and causes 60,000 people to be hospitalised annually. Health care expenditure due to asthma in Australia was $606 million in 2004–2005. There are four primary biological factors that function in the initiation and exacerbation of asthma. Airway inflammation is important as it is often the first response to an airway insult, initiating the three other components: bronchoconstriction, mucus hyper-secretion and hyper-reactivity. The mediators involved in asthma are still not well understood, and current anti-inflammatory corticosteroid treatments are not effective with all asthmatics. As there is currently no cure for asthma, and airway inflammation is the primary component of the disease, it is important that we understand and investigate the mediators of airway inflammation to look for a potential cure and to produce better therapeutics to treat the inflammation. Trefoil factors (TFFs) and secretoglobins (SCGBs) are small secreted proteins involved in the mediation of inflammation and epithelial restitution. TFFs are pro-inflammatory and SCGBs anti-inflammatory by nature. The hypothesis of this study is that in response to induced acute airway inflammation, the expression of TFF1 and TFF3 will increase and expression of SCGB1A1 and SCGB3A2 will decrease in non-asthmatics (N-A), asthmatics medicating with bronchodilators (A-BD) and asthmatics medicating with corticosteroids (A-ST). When comparing the three groups, we expect to see higher expression of the TFFs in the A-BD group compared to the N-A and A-ST groups, indicating that inflammation is mediated by TFFs in asthma and that corticosteroid medication controls their expression as part of the control of inflammation. We expect to see the opposite with SCGBs, with a greater decrease in the A-BD group compared to the other two groups, suggesting that the A-BD group has the least anti-inflammatory activity in response to inflammatory insult. Epigenetic modification plays a role in the regulation of genes that initiate disease states such as inflammatory conditions and cancers. Histone acetylation is one such modification, which involves the acetylation of histones in chromatin by histone acetyltransferases (HATs). This increases the transcription of genes involved with inflammation or enrols histone deacetylases (HDACs) to down-regulate the transcription of inflammatory genes. These HATs and HDACs work in a homeostatic fashion; however, in the event of inflammation, increased HAT activity can stimulate further inflammation, which is believed to be the mechanism involved in some inflammatory diseases. This study hypothesises that in response to inflammation, the expression of HDACs (HDAC1-5) will decrease and the expression of HATs (NCOA1-3, HAT-1 and CREBBP) will increase in all groups. When comparing the expression between the groups, it was expected that a greater decrease in HDACs and a greater increase in HATs will be seen in the A-BD group compared to the other two groups. This would identify histone acetylation as a mechanism involved in the inflammatory condition of asthma and indicate that corticosteroids may treat the inflammation in asthma at least in part by controlling histone acetylation. The aim of the project was to compare the expression of inflammatory genes TFF1, TFF3, SCGB1A1 and SCGB3A2, as well as to compare the gene expression of HDAC1-5, NCOA1-3, HAT-1 and CREBBP within and between N-A (n=15), A-BD (n=15) and A-ST (n=15) groups in response to inflammation. This was performed by collecting airway cells and proteins by sputum induction in three sessions. The sessions were coordinated into an initial baseline collection (SI-1), followed by a second session at least one week later (SI-2) and a third session, six hours after SI-2 to collect a sample containing the resultant acute inflammation caused in SI-2 (SI-3). Analysis of the SI-1 and SI-2 samples in all three groups had high amounts of variability between samples. The samples were taken at least one weak apart and the environmental stimuli on each participant outside of the testing sessions could not be controlled. For this reason, the SI-1 samples were not used for analysis; instead SI-2 and SI-3 samples were compared as they were same-day collections, reducing the probability of differences being due to anything other than the sputum induction. The gene expressions of the TFFs, SCGBs, HDACs and HATs were analysed using real-time PCR. Western blot analysis was performed to analyse the protein concentrations of the TFFs and SCGBs in secreted fractions of the sputum collection. Both the secreted and intracellular protein fractions collected from the sputum inductions for pre- and post-inflammation (SI-2, SI-3) samples of the N-A and A-BD groups were analysed using a proteomic method called iTRAQ. This allowed the comparison of the change in protein expression as a result of airway inflammation in each group. This technique was used as a discovery method to identify novel proteins that are modulated by induced acute airway inflammation. Any proteins of interest would then be further validated and used for future research. Inflammation was achieved in the SI-3 samples of the N-A group with a 21% unit increase in % neutrophils compared to SI-2 (p=0.01). The N-A group had a marked 5.5-fold decrease in HDAC1 gene expression in SI-3 compared to SI-2 (p=0.03). No differences were seen in any of the TFFs, SCGBs or any of the rest of the HDACs and HATs. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed increases in TFF1 and TFF3, and decreases in SCGB1A1 and SCGB3A2 for the majority of SI-3 samples compared to SI-2. The A-BD group also presented a marked increase in neutrophils in the SI-3 samples compared to SI-2 (27% unit increase, p=0.04). The A-BD group had a significant increase in TFF3 and SCGB1A1 gene expression concomitant with induced acute airway inflammation. A 7.3-fold increase in TFF3 (p=0.05) in SI-3 indicated that TFF3 is linked to inflammation in asthmatics. A 2.8-fold increase in SCGB1A1 (p=0.03) indicated that this gene is also up-regulated, suggesting that this SCGB is expressed to try to combat induced acute airway inflammation. No significant changes were seen in any of the other genes analysed. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. However, non-significant analysis of the data displayed an increase in TFF1 and TFF3, and a decrease in SCGB1A1 and SCGB3A2 in SI-3, similar to that seen in the N-A group. The A-ST group was different from the A-BD group, characterised by the use of inhaled corticosteroid medication to treat asthma symptoms. Inhaled corticosteroids are known to treat asthma symptoms through the control of inflammation. Therefore, it was expected that corticosteroid medication would also control the expression of TFFs, SCGBs, HATs and HDACs. Gene expression results only identified a 7.6-fold decrease in HDAC2 expression in SI-3 (p=0.001), which is proposed to be due to the up-regulation of HDAC2 protein that is known to be a function of corticosteroid use. Western blot data did not display any significant changes in the protein levels of the TFFs and SCGBs analysed. The gene expression in SI-2 and SI-3 in each group was compared. When comparing the A-BD group to the N-A group, a 9-fold increase in TFF3 (p=0.008) and a 34-fold increase in SCGB1A1 (p=0.03) were seen in the SI-3 samples. Comparisons of the A-ST group to the N-A group had an increased expression in SI-2 samples for HDAC5 (3.6-fold, p=0.04), NCOA2 (8.5-fold, p=0.04), NCOA3 (17-fold, p=0.01), HAT-1 (36-fold, p=0.003) and CREBBP (13-fold, p=0.001). The SI-3 samples in the A-ST group compared to the N-A group had increased expression for HDAC1 (6.4-fold, p=0.04), HDAC5 (5.2-fold, p=0.008), NCOA2 (9.6-fold, p=0.03), NCOA3 (16-fold, p=0.06), HAT-1 (41-fold, p<0.001) and CREBBP (31-fold, p=0.001). Comparisons of the A-ST group to the A-BD group had SI-2 increases in HDAC1 (3.8-fold, p=0.03), NCOA3 (4.5-fold, p=0.03), HAT-1 (5.3-fold, p=0.01) and CREBBP (23-fold, p=0.001), while SI-3 comparisons saw a decrease in HDAC2 (41-fold, p=0.008) and increases in HAT-1 (4.3-fold, p=0.003) and CREBBP (40-fold, p=0.001). Results showed that TFF3 and SCGB1A1 expression is higher in asthmatics than non-asthmatics and that histone acetylation is more active in the A-ST group than either the N-A or A-BD group, which suggests that histone acetylation activity may be positively correlated with asthma severity. The iTRAQ proteomic analysis of the secreted protein samples identified the SCGB1A1 protein and found it to be decreased in both the N-A and A-BD groups post-inflammation, but significantly so only in the A-BD group. Although no significant results were obtained from the western blot data, both groups displayed a decrease in SCGB1A1 concentration in SI-3 samples, suggesting a correlation with the proteomic data. Only 31 peptides were identified from the secreted samples. The intracellular iTRAQ analysis successfully identified 664 peptides, eight of which had differential expression in association with induced acute airway inflammation. Significant increases were seen in the A-BD group in SI-3 compared to SI-2 than in the N-A group in chloride intracellular channel protein 1, keratin-19, eosinophil cationic protein, calnexin, peroxiredoxin-5, and ATP-synthase delta subunit, while decreases were seen in cystatin-A and mucin-5AC. The iTRAQ analysis was only a discovery measure and further validation must be performed. In summary, the expression of TFFs and SCGBs differed between non-asthmatics and asthmatics. It is clear that TFF3 is active in the airway inflammation associated with asthma as indicated by an increase associated with inflammation in the A-BD group compared to the N-A group. Results for HDAC and HAT genes showed high HAT expression in the A-ST group compared to the N-A and A-BD groups, suggesting that histone acetyltransferases may be responsible for the characteristic unregulated inflammatory symptoms of asthmatics taking corticosteroids. Interestingly, corticosteroid medication did not seem to silence the expression of the analysed HAT genes, which indicates that corticosteroids may not control inflammation by direct regulation of HATs, but instead by competition, most probably with HDAC2 protein. As a discovery tool, iTRAQ is a potent method to both identify and compare the concentration of proteins between samples. The method is a powerful first step into the identification of novel proteins that are regulated in response to different treatments.

Laminin adhesion-selected primary human colon-cancer cells are more tumorigenic than the parenteral and nonadherent cells

Laminin has been shown to promote the malignant phenotype and the level of the 32/67 Kd laminin receptor has been found to correlate with Dukes' staging of colon cancer. A biopsy of a Dukes' stage B2 human colon carcinoma formed a tumor in a nude mouse after coinjection with Matrigel. The parental tumor and the murine tumor appeared identical at the histological level. A cell line LCC-C1 was established from the murine tumor. The cell line appeared moderately differentiated although it did not produce mucin in vitro; however, the xenograft in vivo did produce low levels of mucin. Laminin adherent and non-adherent cell lines were selected. The parental and the laminin-selected cell subclones adhered equally well to plastic and to fibronectin and showed similar growth rates on plastic. When injected subcutaneously into nude mice, the laminin-adherent cells formed relatively undifferentiated tumors that were twice as large as the parental cell tumors whereas the laminin non adherent cells formed very small, but highly differentiated tumors. These data demonstrate that subpopulations of tumor cells which differ in their tumorigenic properties can be selected based on their adhesion to laminin and thus provide models for studying the mechanisms of tumor growth.

Signet-ring cell carcinoma of colorectum : current perspectives and molecular biology

PURPOSE Colorectal signet-ring cell carcinoma (SRCC) is rare, and very little detailed information on the molecular biology of the disease is available. METHODS The literature on the clinical, pathological and, in particular, the molecular biology of this rare entity was critically reviewed. The reviewed articles take into account a total of 1,817 cases of SRCC, but only 143 cases have molecular data available. The characteristics of two patients with colorectal SRCC were also discussed. RESULTS Colorectal SRCC mostly occurs in younger patients, is larger and has different site predilection compared with conventional colorectal adenocarcinoma. It can occur as one of the synchronous cancers in the colorectum. The cancer is usually diagnosed at advanced stages because of the late manifestation of symptoms, and aggressive treatment strategy is required. Limited reports in the literature have shown that the variant of colorectal cancer demonstrated a different pattern of genetic alterations of common growth kinase-related oncogenes (K-ras, BRAF), tumour suppressor genes (p53, p16), gene methylation and cell adhesion-related genes related to the Wingless signalling pathway (E-cadherin and beta-catenin) from conventional colorectal adenocarcinoma. Colorectal SRCC also showed high expression of mucin-related genes and genes related to the gastrointestinal system. There was also a higher prevalence of microsatellite instability-high tumours and low Cox-2 expression in colorectal SRCC as opposed to conventional adenocarcinoma. CONCLUSIONS Colorectal SRCC has unique molecular pathological features. The unique molecular profiles in SRCC may provide molecular-based improvements to patient management in colorectal SRCC.

Oestrogen-modulated gene expression in the human endometrium

To identify key regulatory mechanisms in the growth and development of the human endometrium, microarray analysis was performed on uncultured human endometrium collected during menstruation (M) and the late-proliferative (LATE-P)-phase of the menstrual cycle, as well as after 24 h incubation in the presence of oestradiol (17beta-E2). We demonstrate the expression of novel gene transcripts in the human endometrium. i.e. mucin-9, novel oestrogen-responsive gene transcripts, i.e. gelsolin and flotillin-1, and genes known to be expressed in human endometrium but not yet shown to be oestrogen responsive, i.e. connexin-37 and TFF1/pS2. Genes reported to be expressed during the implantation window and implicated in progesterone action, i.e. secretoglobin family 2A, member 2 (mammaglobin) and homeobox-containing proteins, were up-regulated in uncultured LATE-P-phase endometrium compared to M-phase endometrium. Some gene transcripts are regulated directly by 17beta-E2 alone, others are influenced by the in vivo environment as well. These observations emphasise that the regulation of endometrium maturation by oestrogen entails more then just stimulation of cell proliferation.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Interrelations between Dry Eye Syndrome and Tear Fluid Phospholipid Transfer Protein

The simplified model of human tear fluid (TF) is a three-layered structure composed of a homogenous gel-like layer of hydrated mucins, an aqueous phase, and a lipid-rich outermost layer found in the tear-air interface. It is assumed that amphiphilic phospholipids are found adjacent to the aqueous-mucin layer and externally to this a layer composed of non-polar lipids face the tear-air interface. The lipid layer prevents evaporation of the TF and protects the eye, but excess accumulation of lipids may lead to drying of the corneal epithelium. Thus the lipid layer must be controlled and maintained by some molecular mechanisms. In the circulation, phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) mediate lipid transfers. The aim of this thesis was to investigate the presence and molecular mechanisms of lipid transfer proteins in human TF. The purpose was also to study the role of these proteins in the development of dry eye syndrome (DES). The presence of TF PLTP and CETP was studied by western blotting and mass spectrometry. The concentration of these proteins was determined by ELISA. The activities of the enzymes were determined by specific lipid transfer assays. To study the molecular mechanisms involved in PLTP mediated lipid transfer Langmuir monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) was used. Ocular tissue samples were stained with monoclonal antibodies against PLTP to study the secretion route of PLTP. Heparin-Sepharose affinity chromatography was used for PLTP pull-down experiments and co-eluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. To study whether PLTP plays any functional role in TF PLTP-deficient mice were examined. The activity of PLTP was also studied in dry eye patients. PLTP is a component of normal human TF, whereas CETP is not. TF PLTP concentration was about 2-fold higher than that in human plasma. Inactivation of PLTP by heat treatment or immunoinhibition abolished the phospholipid transfer activity in tear fluid. PLTP was found to be secreted from lacrimal glands. PLTP seems to be surface active and is capable of accepting lipid molecules without the presence of lipid-protein complexes. The active movement of radioactively labeled lipids and high activity form of PLTP to acceptor particles suggested a shuttle model of PLTP-mediated lipid transfer. In this model, PLTP physically transports lipids between the donor and acceptor. Protein-protein interaction assays revealed ocular mucins as PLTP interaction partners in TF. In mice with a full deficiency of functional PLTP enhanced corneal epithelial damage, increased corneal permeability to carboxyfluorescein, and decreased corneal epithelial occludin expression was demonstrated. Increased tear fluid PLTP activity was observed among human DES patients. These results together suggest a scavenger property of TF PLTP: if the corneal epithelium is contaminated by hydrophobic material, PLTP could remove them and transport them to the superficial layer of the TF or, alternatively, transport them through the naso-lacrimal duct. Thus, PLTP might play an integral role in tear lipid trafficking and in the protection of the corneal epithelium. The increased PLTP activity in human DES patients suggests an ocular surface protective role for this lipid transfer protein.

Cloning and expression of zebra fish ferroportin and investigating its influence on anemia

Iron is required for many microbes and pathogens for their survival and proliferation including Leishmania which cause leishmaniasis. Leishmaniasis is an increasingly serious infectious disease with a wide spectrum of clinical manifestations. These range from localized cutaneous leishmaniasis (CL) lesions to a lethal visceral form. Certain strains such as BALB/c mice fail to control L. major infection and develop progressive lesions and systemic disease. These mice are thought to be a model of non-healing forms of the human disease such as kala-azar or diffuse cutaneous leishmaniasis. Progression of disease in BALB/c mice has been associated with the anemia, in last days of their survival, the progressive anemia is considered to be one of the reasons of their death. Ferroportin (Fpn), a key regulator of iron homeostasis is a conserved membrane protein that exports iron across the duodenal enterocytes as well as macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival and proliferation of many microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immune responses and pathogenesis of micoorganisms. To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP–N1. The final resulted plasmid (pEGFP-ZFpn) was used for expression of FPN-EGFP protein in Hek 293T cells. The expression was confirmed by fluorescence microscopy and flow cytometery. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model), NetOGlyc 3.1 server and NetNGlyc 3.1 server. Data emphasised that obtained Fpn from indian zebrafish contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 mucin-type glycosylated amino acid. The results indicate that the prepared and characterized recombinant Fpn protein has no membrane topology difference compared to other Fpn described by other researcher. Our next aim was to deliver recombinant plasmid (pEGFP-ZFpn) to entrocyte cells. However, naked therapeutic genes are rapidly degraded by nucleases, showing poor cellular uptake, nonspecificity to the target cells, and low transfection efficiency. The development of safe and efficient gene carriers is one of the prerequisites for the success of gene therapy. Chitosan and alginate 139 polymers were used for oral gene carrier because of their biodegradability, biocompatibility and their mucoadhesive and permeability-enhancing properties in the gut. Nanoparticles comprising Alginate/Chitosan polymers were prepared by pregel preparation method. The resulting nanoparticles had a loading efficiency of 95% and average size of 188 nm as confirmed by PCS method and SEM images had showed spherical particles. BALB/c mice were divided to three groups. The first and second group were fed with chitosan/alginate nanoparticles containing the pEGFP-ZFpn and pEGFP plasmid, respectively (30 μgr/mice) and the third group (control) didn’t get any nanoparticles. The result showed BALB/c mice infected by L.major, resulted in higher hematocryte and iron level in pEGFP-ZFpn fed mice than that in other groups. Consentration of cytokines determined by ELISA showed lower levels of IL-4 and IL-10 and higher levels of IFN-γ/IL-4 and IFN-γ/IL-10 ratios in pEGFP-ZFpn fed mice than that in other groups. Morover more limited increase of footpad thickness and significant reduction of viable parasites in lymph node was seen in pEGFP-ZFpn fed mice. The results showed the first group exhibited a highr hematocryte and iron compared to the other groups. These data strongly suggests the in vivo administration of chitosan/alginate nanoparticles containing pEGFP-ZFpn suppress Th2 response and may be used to control the leishmaniasis .

Characterization of development-related genes for the cestode Bothriocephalus acheilognathi

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.

姚虻成功从宿主获得血餐的分子机制初探

在长期的吸血进化过程中，吸血节肢动物在唾液腺中形成了一系列有助于适应吸 血生存的活性物质，这些物质包括血管舒张分子、血小板聚集抑制分子、抗凝血分子 和其它相互作用的分子。此外，为了得到洁净的血液和防止在吸血过程中被病原微生 物感染，吸血节肢动物在其唾液腺中形成了许多防御物质以保护自身和宿主，这些物 质包括抗菌肽和蛋白酶抑制剂等。因此，研究吸血节肢动物的唾液腺重要活性物质和 转录体组学有助于弄清其吸血机制。 姚虻（Tabancus yao Macquart）是我国特有的牛虻，其雌性在产卵前需要吸食大 型哺乳动物的血液以促使卵的发育。我们希望通过对姚虻唾液腺重要活性物质和转录 体组学的研究揭示姚虻成功从宿主获得血餐的分子机制，找到具有药用前景的活性物 质和为控制该虫及其传播的疾病的提供理论基础。 首先，我们对其唾液腺匀浆物活性进行了系统分析，发现姚虻唾液腺匀浆物具有 如下活性：有抗金黄色葡萄球菌活性，并且对大肠杆菌、白色念球菌和枯草杆菌都有 效；能够抑制ADP 诱导的洗涤和富血浆血小板的聚集；能够凝集兔红细胞、能够抑制 丝氨酸蛋白酶对小肽底物的水解、具有对纤维蛋白原的水解活性（金属蛋白酶）、具 有过敏原活性、能够促进肥大细胞脱粒和组氨释放；反复检测而没有发现的活性如下： 磷脂酶A2(PLA2)活性、溶血活性、血浆凝固活性、体外抗补体、血管生长促进与抑制 活性、对小鼠脾细胞因子分泌的促进和抑制活性、抗肿瘤细胞HepG2 的生长活性。 以来源姚虻唾液腺的mRNA为材料，我们成功构建了丰度为1ｘ106的姚虻唾液腺 cDNA文库。通过对400个随机克隆的测序，我们得到了编码23种保守蛋白，44种分泌 蛋白和5种功能未知的蛋白。44种分泌蛋白中比较重要的分别是：20种抗原5相关蛋白、 2种α淀粉酶、2种麦芽糖酶、2种attactins抗菌蛋白以及血管舒张肽、过氧化物酶、 抗菌肽、透明质酸酶、mucin样蛋白和脯氨酸丰富蛋白。另外，一些不知道功能的分 泌肽也被发现,这其中包括四个与Hybomitra bimaculata的分泌肽相似性达47-82%的 多肽，这些信息有助于我们发现牛虻唾液腺活性物质和加快从知道部分氨基酸序列的 蛋白的鉴定速度，加快对姚虻从宿主获得血餐的分子机制的诠释速度。 通过分子筛、高压液相色谱等程序，我们从姚虻唾液腺中得到了一个由55 个氨 基酸组成，分子量为6 kDa，含有3 对二硫键的Kunitz 型丝氨基酸蛋白酶抑制剂TYTI。 该抑制剂与Anemonia sulcata 的蛋白酶抑制剂AsKC3 和SA5II 的成熟肽部分的同源 性达66%；并且该抑制剂，对热相对稳定；对凝乳酶、弹性蛋白酶、凝血酶、胰酶等 都有抑制作用，对胰酶的抑制常数为2.586ｘ10-4M 。 通过分子筛、高压液相色谱等程序，我们从姚虻唾液腺中得到了一分子量为7 kDa，由65 个氨基酸组成且含有3 对二硫键的防御素Taymin，它与长角血蜱的防御素的相 似性达43%，但它的第二个半胱氨酸比长角血蜱的半胱氨酸靠前一个氨基酸。该抗菌 肽对大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和白色念球菌的最小抑菌浓度分别是: 160、80、140 和120μg/mL。 通过与分离丝氨酸蛋白酶抑制剂相同的生化分离手段,从姚虻唾液腺中得到一种 红细胞凝集素样活性物质TYML1，其能凝集原始和经胰酶、链霉蛋白酶和弹性蛋白酶 处理的A、B、O 和AB 血型的人、兔、绵羊、大鼠、小鼠、鹌鹑的红细胞, 对链霉蛋 白酶处理的鹌鹑红细胞的凝集效价比正常下降了8 倍；对热、酸、碱处理和蛋白酶降 解稳定；具有Ca2+依赖性，活性能为半乳糖胺和胎球蛋白所完全抑制。 通过分子筛、阴离子交换、高压液相色谱等程序，我们从姚虻唾液腺中分离得到 了一分子量为26 kDa,由234 个氨基酸组成，含有10 个半胱氨酸的血小板聚集抑制剂 Macquaritin-2,它与报道的所有血小板聚集抑制剂均不具有同源性，但是与双翅目昆 虫唾液腺过敏原却有一定的同源性(25%-33%),对其血小板聚集抑制活性研究发现:其 能抑制胰酶、花生四烯酸、Stejnulxin、TMVA、ADP、U46619 等激动剂诱导的血小板 聚集；血小板膜结合试验表明:其能与血小板细胞膜结合,故该血小板聚集抑制剂可能 通过作用血小板上的受体来阻止激动剂诱导血小板聚集。 通过分子筛、阳离子交换、高压液相色谱等程序，我们从姚虻唾液腺中分离得到 分子量为24-30 kDa 的两个血小板聚集抑制剂Macquaritin-3 和Macquaritin-1，它 们的N 端16 个氨基酸分别是V N Y C R L P C R G C D Y H V 和 V A V D Y L G L P G R G Y H V。通过PCR, Macquaritin-3 的核苷酸序列被得到，其推导蛋白的成熟区 和信号肽分别含有232 和23 个氨基酸。利用根据Macquaritin-1 的N 端氨基酸设计 的简并引物扩到含有V A V D Y L G L P 序列的两组核苷酸序列。它们与报道的所有 血小板聚集抑制剂均不具有同源性，但是其与双翅目昆虫的唾液腺抗原5 相关蛋白却 有一定的同源性。将所有血小板抑制剂及其相关序列和通过cDNA 文库筛选得到的抗 原5 相关序列进行分析,发现它们有很高的相似性,这种相似性介于33.3%-93.0%间, 且大部分高于50%。另外，两个血小板聚集抑制剂和一个可能的血小板抑制剂分别处 于这些抗原5 相关蛋白进化树的三个簇中。因此，我们推测这些过抗原5 相关蛋白可 能都具有血小板抑制剂活性。

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the. presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml(-1)). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. All rights reserved.

Metabolism of host-derived carbohydrates by Bifidobacterium breve UCC2003

Bifidobacteria are Gram positive, anaerobic, typically Y-shaped bacteria which are naturally found in the digestive tract of certain mammals, birds and insects. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breast-fed infants. The prototypical B. breve strain UCC2003 has previously been shown to utilise numerous carbohydrates of plant origin. Various aspects of host-derived carbohydrate metabolism occurring in this bacterium will be described in this thesis. Chapter II describes B. breve UCC2003 utilisation of sialic acid, a nine-carbon monosaccharide, which is found in human milk oligosaccharides (HMOs) and the mucin glycoprotein. B. breve UCC2003 was also shown to cross-feed on sialic acid released from 3’ sialyllactose, a prominent HMO, by the extracellular sialidase activity of Bifidobacterium bifidum PRL2010. Chapter III reports on the transcriptional regulation of sialic acid metabolism in B. breve UCC2003 by a transcriptional repressor encoded by the nanR gene. NanR belongs to the GntR-family of transcriptional regulators and represents the first bifidobacterial member of this family to be characterised. Chapter IV investigates B. breve UCC2003 utilisation of mucin. B. breve UCC2003 was shown to be incapable of degrading mucin; however when grown in co-culture with B. bifidum PRL2010 it exhibits enhanced growth and survival properties. A number of methods were used to investigate and identify the mucin components supporting this enhanced growth/viability phenotype. Chapter V describes the characterisation of two sulfatase-encoding gene clusters from B. breve UCC2003. The transcriptional regulation of both sulfatase-encoding gene clusters was also investigated. The work presented in this thesis represents new information on the metabolism of host-derived carbohydrates in bifidobacteria, thus increasing our understanding of how these gut commensals are able to colonise and persist in the gastrointestinal tract.

Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease

This study describes the formulation, characterisation and preliminary clinical evaluation of mucoadhesive, semi-solid formulations containing hydroxyethylcellulose (HEC, 1-5%, w/w), polyvinylpyrrolidine (PVP, 2 or 3%, w/w), poly carbophil (PC, 1 or 3%, w/w) and tetracycline (5%, w/w, as the hydrochloride). Each formulation was characterised in terms of drug release, hardness, compressibility, adhesiveness (using a texture analyser in texture profile analysis mode), syringeability (using a texture analyser in compression mode) and adhesion to a mucin disc (measured as a detachment force using the texture analyser in tensile mode). The release exponent for the formulations ranged from 0.78+/-0.02 to 1.27+/-0.07, indicating that drug release was non-diffusion controlled. Increasing the concentrations of each polymeric component significantly increased the time required for 10 and 30% release of the original mass of tetracycline, due to both increased viscosity and, additionally, the unique swelling properties of the formulations. Increasing concentrations of each polymeric component also increased the hardness, compressibility, adhesiveness, syringeability and mucoadhesion of the formulations. The effects on product hardness, compressibility and syringeability may be due to increased product viscosity and, hence, increased resistance to compression. Similarly, the effects of these polymers on adhesiveness/mucoadhesion highlight their mucoadhesive nature and, importantly, the effects of polymer state (particularly PC) on these properties. Thus, in formulations where the neutralisation of PC was maximally suppressed, adhesiveness and mucoadhesion were also maximal. Interestingly, statistical interactions were primarily observed between the effects of HEC and PC on drug release, mechanical and mucoadhesive properties. These were explained by the effects of HEC on the physical state of PC, namely swollen or unswollen. In the preliminary clinical evaluation, a formulation was selected that offered an appropriate balance of the above physical properties and contained 3% HEC, 3% PVP and 1% PC, in addition to tetracycline 5% (as the hydrochloride). The clinical efficacy of this (test) formulation was compared to an identical tetracycline-devoid (control) formulation in nine periodontal pockets (greater than or equal to 5 mm depth). One week following administration of the test formulation, there was a significant improvement in periodontal health as identified by reduced numbers of sub-gingival microbial pathogens. Therefore, it can be concluded that, when used in combination with mechanical plaque removal, the tetracycline-containing semi-solid systems described in this study would augment such therapy by enhancing the removal of pathogens, thus improving periodontal health. (C) 2000 Elsevier Science B.V. All rights reserved.