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Pancreatic cancer (PC) remains the fourth leading cause of cancer death; therefore, there is a clinically unmet need for novel therapeutics and diagnostic markers to treat this devastating disease. Physicians often rely on biopsy or CT for diagnosis, but more specific protein biomarkers are highly desired to assess the stage and severity of PC in a noninvasive manner. Serum biomarkers such as carbohydrate antigen 19-9 are of particular interest as they are commonly elevated in PC but have exhibited suboptimal performance in the clinic. MUC5AC has emerged as a useful serum biomarker that is specific for PC versus inflammation. We developed RA96, an anti-MUC5AC antibody, to gauge its utility in PC diagnosis through immunohistochemical analysis and whole-body PET in PC. Methods: In this study, extensive biochemical characterization determined MUC5AC as the antigen for RA96. We then determined the utility of RA96 for MUC5AC immunohistochemistry on clinical PC and preclinical PC. Finally, we radiolabeled RA96 with (89)Zr to assess its application as a whole-body PET radiotracer for MUC5AC quantification in PC. Results: Immunohistochemical staining with RA96 distinguished chronic pancreatitis, pancreatic intraepithelial neoplasia, and varying grades of pancreatic ductal adenocarcinoma in clinical samples. (89)Zr-desferrioxamine-RA96 was able to detect MUC5AC with high specificity in mice bearing capan-2 xenografts. Conclusion: Our study demonstrated that RA96 can differentiate between inflammation and PC, improving the fidelity of PC diagnosis. Our immuno-PET tracer (89)Zr-desferrioxamine-RA96 shows specific detection of MUC5AC-positive tumors in vivo, highlighting the utility of MUC5AC targeting for diagnosis of PC.

Cervical mucus plays an important role in female fertility, since it allows the entry of motile and morphological normal sperm while preventing the ascent of pathogens from the vagina. The function of cervical mucus is critically linked to its rheological properties that are in turn dictated by O-glycosylated proteins, called mucins. We aimed to characterize the O-glycan composition in the cervical mucus of six European ewe breeds with known differences in pregnancy rates following cervical/vaginal artificial insemination with frozen–thawed semen, which are due to reported differences in cervical sperm transport. These were Suffolk (low fertility) and Belclare (medium fertility) in Ireland, Ile de France and Romanov (both with medium fertility) in France, and Norwegian White Sheep (NWS) and Fur (both with high fertility) in Norway (n = 28–30 ewes/breed). We identified 124 O-glycans, from which 51 were the major glycans with core 2 and fucosylated glycans as the most common structures. The use of exogenous hormones for synchronization did not affect the O-glycan composition in both high-fertility ewe breeds, but it did in the other four ewe breeds. There was a higher abundance of the sulfated glycan (Galβ1–3[SO3-GlcNAcβ1–6]GalNAc), fucosylated glycan (GlcNAcβ1–3(Fucα1–2Galβ1–3)GalNAc) and core 4 glycan (GlcNAcβ1–3[GlcNAcβ1–6]GalNAc) in the low-fertility Suffolk breed compared with NWS (high fertility). In addition, core 4 glycans were negatively correlated with mucus viscosity. This novel study has identified O-glycans that are important for cervical sperm transport and could have applications across a range of species including human.

Sialic acid (Sia) is a major constituent of both the sperm glycocalyx and female reproductive mucosal surface and is involved in regulating sperm migration, uterotubal reservoir formation and oocyte binding. Siglecs (sialic acid-binding immunoglobulin – like lectins) commonly found on immune cells, bind to Sia in a linkage- and sugar-specific manner and often mediate cell-to-cell interactions and signalling. Proteomic and transcriptomic analysis of human and bovine sperm have listed Siglecs, but to date, their presence and/or localisation on sperm has not been studied. Therefore, the aim of this study was to characterise the presence of Siglecs on the surface of bovine, human and ovine sperm using both immunostaining and Western blotting. Siglec 1, 2, 5, 6, 10 and 14 were identified and displayed both species- and regional-specific expression on sperm. Almost universal expression across Siglecs and species was evident in the sperm neck and midpiece region while variable expression among Siglecs, similar among species, was detected in the head and tail regions of the sperm. The possible role for these proteins on sperm is discussed.

There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions.

BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a small conductance adenosine 3',5'-cyclic monophosphate (cAMP)-activated chloride ion channel found in the apical membranes of epithelia within the pancreas, airway, intestine, bile duct, sweat gland, and male genital ducts. Pancreatic insufficiency is a feature of about 85% of patients with cystic fibrosis and is believed to be caused by pancreatic autolysis after pancreatic duct obstruction. The aim of this study was to investigate the expression of CFTR in the pancreas from early development to postnatal life to establish whether the CFTR plays a key role in development of the pancreatic duct epithelium. METHODS: Expression of CFTR from the start of the mid- trimester of human development through term to adult life by messenger RNA (mRNA) in situ hybridization was examined. RESULTS: CFTR mRNA is detected throughout the pancreatic duct epithelium and its pattern of expression follows the differentiation of the duct system. CONCLUSIONS: CFTR is a valuable marker of human pancreatic duct cell development and differentiation. (Gastroenterology 1997 Sep;113(3):914-9)

In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B).

In Rhizobium leguminosarum both the C4-dicarboxylate transport system and wild-type lipopolysaccharide layer (LPS) are essential for nitrogen fixation. A Tn5 mutant (RU301) of R. leguminosarum bv. viciae 3841, was isolated that is only able to synthesize LPS II, which lacks the O-antigen. Strain RU301 exhibits a rough colony morphology, flocculates in culture and is unable to swarm in TY agar. It also fails to grow on organic acids, sugars or TY unless the concentration of calcium or magnesium is elevated above that normally required for growth. The defects in the LPS and growth in strain RU301 were complemented by a series of cosmids from a strain 3841 cosmid library (pRU3020-pRU3022) and a cosmid from R. leguminosarum bv. phaseoli 8002 (pIJ1848). The transposon insertion in strain RU301 was shown to be located in a 3 kb EcoRI fragment by Southern blotting and cloning from the chromosome. Sub-cloning of pIJ1848 demonstrated that the gene disrupted by the transposon in strain RU301 is located on a 2.4 kb EcoRI-PstI fragment (pRU74). R. leguminosarum bv. viciae VF39-C86, which is one of four LPS mutants previously isolated by U. B. Priefer (1989, J Bacteriol 171, 6161-6168), was also complemented by sub-clones of pIJ1848 but not by pRU74, suggesting the mutation is in a gene adjacent to that disrupted in strain RU301. Complementation and Southern analysis indicate that the region contained in pIJ1848 does not correspond to any other cloned Ips genes. Two dctA mutants, RU436 and RU437, were also complemented by pIJ1848 and pRU3020. Mapping of pIJ1848 and Southern blotting of plasmid-deleted strains of R. leguminosarum revealed that dctD and the region mutated in strain RU301 are located approximately 10 kb apart on the chromosome. Analysis of homogenotes demonstrated that there is not a large region important in calcium utilization, organic acid metabolism or LPS biosynthesis located between the gene disrupted in strain RU301 and dctD. Strain VF39C-86 also required an elevated concentration of calcium for growth on succinate, while strains mutated in the alpha-chromosomal or beta-plasmid group of Ips genes grew at the same calcium concentrations as the wild type, demonstrating that the additional calcium requirement is not a property of all LPS rough mutants. Strain RU301 nodulates peas, but does not reduce acetylene, demonstrating that the gene mutated in this strain is essential for nitrogen fixation.

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.

Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.