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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.

Molecular manipulation and expression of mucins, large glycoproteins that provide the structural framework of mucus, are challenging due to mucins' size and numerous domains, including variable number tandem repeat (VNTRs) regions that are sites of O-glycosylation. Only individual human mucin domains have been expressed in mammalian cells. We produced recombinant versions of MUC5AC, a major secreted mucin in the respiratory tract, encoding the N-terminus, C-terminus, N- and C-termini together, and N- and C-termini interspersed with two native tandem repeat sequences (N+2TR+C) in both tracheal and bronchial cell lines. The latter protein contains all of the functional domains required for the biosynthesis and secretion of glycosylated mucin. The N-terminus protein was found in monomeric and higher molecular mass forms suggesting that secreted MUC5AC may form a branched netlike structure analogous to that described for MUC2. At the C-terminus, proteins underwent cleavage, polymerization, and glycosylation. Thus, they appear to undergo pivotal processing steps as predicted for native MUC5AC, which is analogous to that for other individual recombinant mucin domains. Secretion occurred when cells were grown on transwell filter inserts but not on plastic, indicating that the extracellular environment likely plays a role in mucin processing. The secreted N+2TR+C protein differed in molecular mass from the intracellular form, indicating that additional processing occurred. These recombinant proteins, expressed in different backgrounds, can potentially address the role of different mucin domains on MUC5AC processing and function as well as the role of MUC5AC in health and disease.

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.

This study tested the hypothesis that sperm sialic acid (Sia) is required to reach the site of fertilization, and that successful fertilization requires recognition of Sia from both the sperm and oocyte to occur. In addition, it has recently been reported that Siglecs (Sia-binding-immunoglobulin-like lectins) are present on the sperm surface. Thus, the possibility that the recognition of oocyte Sia was sperm-Siglec-mediated was also addressed. Sperm exposed to neuraminidase (NMase) exhibited lower overall and progressive motility, which translated to a decreased ability to swim through cervical mucus from cows in oestrus. In addition, when either sperm or cumulus–oocyte complexes (COCs) were treated with NMase, a decrease in cleavage and blastocyst rate was observed. However, incubation of sperm with increasing concentrations of anti-Siglec-2, -5, -6 and -10 antibodies prior to fertilization had no effect on their fertilizing ability. Interestingly, treatment with NMase increased the number of sperm bound to the ZP but also the rate of polyspermic fertilization. Flow cytometry analysis revealed no differences in the percentage of capacitated or acrosome-reacted sperm. These results suggest that Sia are required to reach the site of fertilization but need to be removed for sperm–oocyte interaction. However, fine regulation is needed to avoid abnormal fertilization which can lead to impaired embryo development.

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.

The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of β-galactosidase, α-L-fucosidase, β-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of β-galactosidase and sialidase was observed 12 h after onset of estrus, whereas β-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.

The commercial applicability of bovine artificial insemination (AI) depends on the effectiveness of diluents for maintaining sperm fertility. Challenges faced by the AI industry due to recent advances in assisted reproduction, and the limitations inherent in using fresh and frozen-thawed sperm for AI, could be overcome with the development of better semen diluents. Research into the different microenvironments of bovine sperm as they progress towards maturity, capacitation and fertilisation is revealing various mechanisms that could be exploited to improve the formulation of semen diluents. These are reviewed here. A rationale for a more detailed investigation of bovine cervical mucus for factors that may allow further progress towards this goal are also discussed.

The biochemical and biophysical properties of mucins are largely determined by extensive O-glycosylation of serine- and threonine-rich tandem repeat (TR) domains. In a number of human diseases aberrant O-glycosylation is associated with variations in the properties of the cell surface-associated and secreted mucins. To evaluate in vivo the O-glycosylation of mucin TR domains, we generated recombinant chimeric mucins with TR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted for the native TRs of epitope-tagged MUC1 protein (MUC1F). These hybrid mucins were extensively O-glycosylated and showed the expected association with the cell surface and release into culture media. The presence of different TR domains within the chimeric mucins appears to have limited influence on their posttranslational processing. Alterations in glycosylation were detailed by fast atom bombardment mass spectrometry and reactivity with antibodies against particular blood-group and tumor-associated carbohydrate antigens. Future applications of these chimeras will include investigations of mucin posttranslational modification in the context of disease.

The malate dehydrogenase gene, mdh, from Rhizobium leguminosarum encodes a protein with a molecular weight of 33 590 that associates as a homotetramer. It is a lactate dehydrogenase-like malate dehydrogenase that most closely resembles the protein from the colonial green alga Botryococcus braunii. Malate dehydrogenase from R. leguminosarum has a Km of 74 WM and a Vmax of 822 Wmol min 31 mg 31 protein for oxaloacetate, while the Km for malate is 3.6 mM and the Vmax 62 Wmol min 31 mg 31 protein. Downstream of mdh are sucCDAB, which encode succinyl-CoA synthetase (sucCD) and the E1 and E2 components of the K-ketoglutarate dehydrogenase complex (sucAB). Complementation and LacZ fusion analysis indicates that there is a common promoter for the mdh-suc genes. Mutation of downstream genes in the mdh-suc operon increases transcription 7-fold from the mdh promoter. The transcriptional coupling of mdh and sucCDAB is likely to be important in the regulation of carbon and nitrogen metabolism in bacteroids. fl 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

The dctA gene, coding for the dicarboxylate transport protein, has an inducible promoter dependent on activation by the two-component sensor-regulator pair DctB and DctD. LacZ fusion analysis indicates that there is a single promoter for dctB and dctD . The dctA promoter is also induced by nitrogen limitation, an effect that requires DctB-DctD and NtrC. DctB alone is able to detect dicarboxylates in the absence of DctA and initiate transcription via DctD. However, DctA modifies signal detection by DctB such that in the absence of DctA, the ligand specificity of DctB is broader. dctAp also responds to heterologous induction by osmotic stress in the absence of DctA. This effect requires both DctB and DctD. A transposon insertion in the dctA-dctB intergenic region ( dctA101 ) which locks transcription of dctA at a constitutive level independent of DctB-DctD results in improper signalling by DctB-DctD. Strain RU150, which carries this insertion, is defective in nitrogen fixation (Fix − ) and grows very poorly on ammonia as a nitrogen source whenever the DctB-DctD signalling circuit is activated by the presence of a dicarboxylate ligand. Mutation of dctB or dctD in strain RU150 reinstates normal growth on dicarboxylates. This suggests that DctD-P improperly regulates a heterologous nitrogen-sensing operon. Increased expression of DctA, either via a plasmid or by chromosomal duplication, restores control of DctB-DctD and allows strain RU150 to grow on ammonia in the presence of a dicarboxylate. Thus, while DctB is a sensor for dicarboxylates in its own right, it is regulated by DctA. The absence of DctA allows DctB and DctD to become promiscuous with regard to signal detection and cross talk with other operons. This indicates that DctA contributes significantly to the signalling specificity of DctB-DctD and attenuates cross talk with other operons.

Background and aims—Mucin glycoproteins play a key role in the normal function of the epithelium lining the gastrointestinal tract. The expression of mucin genes, MUC 3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues was examined to establish the localisation and age of onset of expression of each mucin gene during human development.Methods—Mucin gene expression was assayed by mRNA in situ hybridisation.Results—Expression of MUC3 was detected in the small intestine and colon from 13 weeks gestation onwards and at low levels in the main pancreatic duct at 13 weeks only. MUC4 expression was seen at a low level in the colonic epithelium from 13 weeks of gestation but not elsewhere in the gastrointestinal tract. MUC5AC mRNA was detected in the colon at 17 weeks and at high levels in the stomach at 23 weeks. MUC6transcripts were evident in the pancreatic ducts from 13 weeks of gestation and at high levels in the stomach at 23 weeks.MUC5B, MUC7, and MUC8transcripts were not detected.Conclusions—Mucin genes are expressed from the early mid-trimester of gestation in the developing human fetal gastrointestinal tract.

Mucin glycoproteins play a key role in the normal function of the airway epithelium. We examined the expression of mucin genes, MUC3, 4, 5AC, 5B, 6, 7, and 8 in human fetal tissues to establish the localization and age of onset of expression of each mucin gene during human development. We detected expression of MUC4, 5AC, 5B, and 7 in the mid-trimester airway epithelium but did not detect expression of MUC3, 6, or 8. MUC4 was expressed in the trachea and large airways in the majority of cells in the airway epithelium. Expression of MUC5AC was only seen in individual goblet cells in the trachea, while MUC5B was expressed in the surface epithelium of the trachea at 13 wk but was largely restricted to submucosal glands by 23 wk of gestation.

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)

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.

Mucus within the cervical canal represents a hormonally regulated barrier that reconciles the need to exclude the vaginal microflora from the uterus during progesterone dominance, while permitting sperm transport at estrus. Its characteristics change during the estrous cycle to facilitate these competing functional requirements. Hydrated mucin glycoproteins synthesized by the endocervical epithelium form the molecular scaffold of this mucus. This study uses the bovine cervix as a model to examine functional groups of genes related to mucin biosynthesis and mucus production over the periestrous period when functional changes in cervical barrier function are most prominent. Cervical tissue samples were collected from 30 estrus synchronized beef heifers. Animals were slaughtered in groups starting 12 h after the withdrawal of intravaginal progesterone releasing devices (controlled internal drug releases) until 7 days postonset of estrus (luteal phase). Subsequent groupings represented proestrus, early estrus, late estrus, metestrus, and finally the early luteal phase. Tissues were submitted to next generation RNA-seq transcriptome analysis. We identified 114 genes associated with biosynthesis and intracellular transport of mucins, and postsecretory modifications of cervical; 53 of these genes showed at least a twofold change in one or more experimental group in relation to onset of estrus, and the differences between groups were significant ( P < 0.05). The majority of these genes showed the greatest alteration in their expression in the 48 h postestrus and luteal phase groups.

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.

On the basis of enzyme assays, myo-inositol appears to be catabolized via 2-keto-myo-inositol and D-2,3-diketo-4-deoxy epi-inositol in Rhizobium leguminosarum bv. viciae, as occurs in Klebsiella aerogenes. The first two enzymes of the pathway, myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase were increased 7- and 77-fold, respectively, after growth of R. leguminosarum on myo-inositol compared to glucose. Cells of R. leguminosarum grown on glucose as the carbon source and then resuspended in myo-inositol, increased myo-inositol-dependent O2 consumption by sixfold in 3 h, confirming this to be an inducible pathway. Succinate, malate and glucose exhibited strong catabolite repression of the myo-inositol catabolic pathway with myo-inositol dehydrogenase and 2-keto-myo-inositol dehydratase being repressed by at least 68% and 80%, respectively, in all cases. A dicarboxylate transport mutant (dctA) was unable to repress either enzyme when grown on myo-inositol and succinate. There was no repression of the first two enzymes of the myo-inositol catabolic pathway in the background of constitutive expression of the dicarboxylate transport system, indicating a dicarboxylate must be present to cause repression. The data imply that dicarboxylates are required intra-cellularly to repress these enzymes. Three transposon mutants were isolated that cannot grow on myo-inositol as the sole carbon source and were unable to induce either myo-inositol dehydrogenase or 2-keto-myo-inositol dehydratase. The mutants were unaffected in the ability to nodulate peas and vetch. Furthermore, vetch plants infected with one mutant (RU360) reduced acetylene at the same rate as plants infected with the wild type. Bacteroids did not oxidize myo-inositol, nor were the catabolic enzymes detected, confirming myo-inositol is not important as an energy source in bacteroids. The possible role of myo-inositol in the rhizosphere is considered.

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.

The MUC 6 mucin cDNA was isolated from a human stomach cDNA library and has been shown to be expressed in a number of other tissues in the gastrointestinal tract, including the gallbladder, pancreas, and parts of the ileum and colon. Here we establish that MUC 6 is expressed transiently in the nephrogenic zone of the kidney in the early mid-trimester of development. MUC 6 transcripts were detected in the epithelium of ureteric buds at 13 weeks and at lower levels from 17 to 23 weeks of gestation. Traces of MUC 6 mRNA were seen in the collecting ducts but not elsewhere in the developing kidney, and MUC 6 glycoprotein was detected in the epithelium of ureteric buds and collecting ducts. MUC 6 transcripts were absent from adult kidney. This pattern of expression of MUC 6 in the developing kidney suggests a role in epithelial organogenesis. MUC 6 transcripts were also present at low levels in mid-trimester epididymal epithelium.

2To whom correspondence should be addressed The cause of the mucus clearance problems associated with cystic fibrosis remains poorly understood though it has been suggested that mucin hypersecretion, dehydration of mucins, and biochemical abnormalities in the glycosylation of mucins may be responsible. Since the biochemical and biophysical properties of a mucin are dependent on O-glycosylation, our aim was to evaluate the O-glycosylation of a single mucin gene product in matched pairs of cells that differed with respect to CFTR expression. An epitope-tagged MUC1 mucin cDNA (MUC1F) was used to detect variation in mucin glycosylation in stably transfected colon carcinoma cell lines HT29 and Caco2. The glycosylation of MUC1F mucin was evaluated in matched pairs of Caco2 cell lines that either express wild-type CFTR or have spontaneously lost CFTR expression. The general glycosylation pattern of MUC1F was evaluated by determining its reactivity with a series of monoclonal antibodies against known blood group and tumor-associated carbohydrate antigens. Metabolic labeling experiments were used to estimate the gross levels of glycosylation and sulfation of MUC1F mucin in these matched pairs of cell lines. Expression of CFTR in this experimental system did not affect the gross levels of glycosylation or sulfation of the MUC1F mucin nor the types of carbohydrates structures attached to the MUC1F protein.

MUC1 is expressed at the apical surface of ductal epithelia of tissues, including breast, pancreas, airway, and the gastrointestinal tract, where its functions include lubrication and protection of the epithelia. In addition, roles for MUC1 have been suggested in both adhesive and antiadhesive properties of tumor cells, and extensive O-glycosylation of the MUC1 tandem repeat domain may contribute to these functions. Little information is available on the specific O-glycosylation of MUC1. One problem in identifying different MUC1 glycoforms has been that monoclonal antibodies raised against the MUC1 core protein recognize epitopes in the tandem repeat domain, which is often glycosylated to an extent that obscures these epitopes. We developed an epitope-tagged form of MUC1 that allowed the detection of multiple MUC1 glycoforms and established the presence of a number of important blood group and tumor-associated carbohydrate antigens on MUC1 expressed by two pancreatic tumor cell lines (Panc-1 and S2-013) and two colon tumor cell lines (Caco-2 and HT-29). Antigens detected include sialyl-Lewisa, sialyl-Lewisc, sialyl-Lewisx, and sialyl-Tn.

Cosmid-borne and chromosomal lacZ fusions to aapJ, aapQ and aapM were used to examine the nitrogen regulation of the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841. Transcription of the first gene of the operon (aapJ), which encodes the periplasmic binding protein, was 2–4-fold higher than aapQ and aapM, which encode the integral membrane proteins, under various growth conditions. This may be due to the presence of a putative stem loop in the intergenic region between aapJ and aapQ. All aap fusions were derepressed 3–5-fold after growth on glutamate as a nitrogen source, which effectively causes nitrogen limitation. An ntrC mutant was derepressed for transcription of the aap operon and had high rates of amino acid transport when grown on ammonia as the nitrogen source. Thus NtrC negatively regulates the aap operon, contrary to its usual role in positive gene activation. These results confirm that the aap operon is subject to complex regulation involving both transcriptional and post-transcriptional factors.

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.