Haemonchus contortus glycoproteins contain N-linked oligosaccharides with novel highly fucosylated core structures

Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein.     

Two-dimensional elution map of GalNAc-containing N-linked oligosaccharides

We have previously published a two-dimensional (2-D) mapping technique for N-linked oligosaccharides using pyridylaminated derivatives (PA-oligosaccharides) (N. Tomiya et al. Anal. Biochem. 171, 73-90, 1988). We now report an extension of this method to GalNAc-containing N-linked oligosaccharides. The new 2-D map was prepared from the elution data of 40 different GalNAc-containing oligosaccharides, 16 of which were obtained directly from human urinary kallidinogenase by digestion with glycopeptidase A. The other 24 oligosaccharides were derived by subsequent digestion of the 16 original oligosaccharides with beta-galactosidase or alpha-fucosidase. Each of the 40 oligosaccharide derivatives was separated by high-performance liquid chromatography using ODS-silica and amide-silica columns. The 2-D map constructed by plotting elution position of each oligosaccharide (expressed in terms of glucose units) can be useful as such in delineating the structure of an unknown oligosaccharide by direct placement of its elution positions in the 2-D map. Multiple regression analysis of the data as performed previously yielded parameters related to the contribution of each component monosaccharide unit to the elution profile. The best results were obtained when the GalNAc-containing PA-oligosaccharides were classified into an F-series (those containing Fuc alpha 6GlcNAc-PA) and a Z-series (all others), based on our previous classification method. These calculated values are useful in predicting oligosaccharide structure from known elution values as well as to predict elution volumn from a known structure. The structure of a minor GalNAc-containing oligosaccharide in human urinary kallidinogenase was elucidated using these newly calculated values.     

Tuberculosis of the parotid gland

The presentation of tuberculosis as an isolated parotid lump is rare. In this paper, six cases with tuberculous parotitis are reported which were evaluated as a benign parotid neoplasm in 216 specimens pre-operatively. All but one of them had no previous history of tuberculosis and all had a parotid lump as a sole symptom for at least one year. The diagnosis of tuberculosis was made, after superficial parotidectomy, by histopathology. Parenchymal involvement and intraparotid lymph node involvement with tuberculosis were seen in five and three patients, respectively. Two of the patients had lymph node involvement outside the parotid area. One of six patients had a coincidental Warthin tumour. A surgical approach is not only therapeutic but also diagnostic when other diagnostic tools fail.     

Tumors of the parotid gland

Tumors of the parotid are the most frequently encountered salivary gland tumors. Knowledge of the histology and anatomy of the salivary gland is important when considering the histiogenesis of salivary gland tumors, requiring close cooperation between the pathologist and the surgeon. Most tumors are benign epithelial formations. Pleomorphous adenomas predominate. Superficial lobectomy is adequate treatment. When the tumor involves a deep lobe, total parotidectomy is indicated. Treatment of malignant tumors depends on the histology, its TNM stage and other factors. Total parotidectomy with lymphadectomy and radiotherapy are needed in case of high grade malignancy. In children, vascular neoplasias are the most frequent, followed by malignant tumors. Their histological features and treatment are the same as for adults.     

Recurrences from malignant parotid salivary gland tumors

The clinical course of 130 patients treated for malignant parotid tumors at the three institutions have been reviewed. Fifty-six of these 130 patients developed recurrences following their primary treatment by a surgical procedure. There were a total of 109 recurrences among these 56 patients. The average number of recurrences was two per patient. The average survival from first recurrence was 3.7 years, with the median survival 2 years. The range of survival was 0.5 to 17 years. Once recurrence developed, treatment was by surgery, radiation therapy, chemotherapy, or a combination. Of 56 patients with recurrence, 33 are dead and 9 patients are alive with disease. Fourteen patients are alive and well with no evidence of disease. These NED patients had an average number of 1.6 recurrences and a median survival to date of only 3 years. Our study indicates that for the majority of patients who develop recurrence, survival is relatively short and treatment is usually ineffective in three of four patients. The authors conclude that after a surgical procedure for malignant salivary gland tumors, a trial of wide field postoperative radiation therapy to high dose should be considered as part of the initial treatment.     

Mass spectrometric mapping and sequencing of N-linked oligosaccharides derived from submicrogram amounts of glycoproteins

A size-selected genomic library from Elymus alaskanus was screened for the presence of (GA)n, (GT)n, (CAC)n, and (TCT)n microsatellite sequences. A total of 28 positive clones were found for the two dinucleotide repeats, whereas no positive clones were found for the trinucleotide repeats. Positive clones were sequenced to validate the presence of microsatellites and to generate polymerase chain reaction (PCR) primers, based on the sequences flanking the microsatellite. Primer pairs were designed and evaluated for 18 selected microsatellites. The resulting loci were analysed by PCR for their usefulness as molecular markers in an array of 18 accessions representing E. alaskanus and 10 other Elymus species. PCR amplification revealed alleles for the 18 loci, which varied in having 1-10 alleles in these accessions. In the 18 accessions tested, 7 of the 18 loci were polymorphic, with gene diversity values ranging from 0.54 to 0.80 among all species. Within E. alaskanus, gene diversity values ranged from 0.20 to 0.72, with a mean of 0.48. Polymorphism was also detected within accessions. No clear relationship between total repeat length and the degree of polymorphism was observed in this study. Primer pairs designed to amplify microsatellites in E. alaskanus can be used to generate polymorphism products in other species within the genus. Hence, microsatellites are useful markers for studying both inter- and intra-specific genetic variability within Elymus.     

Epithelial-myoepithelial carcinoma of the parotid gland

We sought to determine if patients with cystic fibrosis and sputum cultures positive for Mycobacterium avium complex (MAC) have delayed-type hypersensitivity to an M. avium sensitin. Seventeen (33%) of 51 selected patients had MAC isolated from at least one sputum culture. Skin tests with purified protein derivative and M. avium sensitin demonstrated that five (10%) of 51 patients were anergic, and anergy was correlated with use of systemic steroids. Sixteen (35%) of 46 nonanergic patients had M. avium-dominant skin test reactions. Twelve (75%) of these 16 patients with cultures positive for MAC had M. avium-dominant skin tests; the specificity of skin testing was 87%. These data suggest that most patients with cystic fibrosis and sputum cultures positive for MAC have infection rather than colonization with MAC. Skin testing with M. avium sensitin is a sensitive and specific method for screening these infections.     

The ultrastructure of the sheep parotid gland

The sheep parotid is a compound tubular gland; its ultrastructure reflects the function of this gland to secrete large amounts of fluid with very little protein. The cells of the secretory tubules possess extensively folded lateral plasma membranes and a fairly large number of mitochondria. Rapid equilibration of water across the epithelium is assured by the close proximity over large areas of intercellular spaces and the wide secretion canaliculi. Numerous long microvilli extend into the latter. Although secretion granules may be quite numerous, there is evidence that many of these granules are not discharged but undergo degradation by lysosomal enzymes. The intercalated ducts are often dilated but excessive distension is probably prevented by bundles of microfilaments in their epithelial cells.     

A megalith of the parotid salivary gland

We present a rare case of a giant intra-parotid calculus in a 64-year-old man with recurrent parotitis. The literature is also reviewed.     

Structural characterisation of N-linked and O-linked oligosaccharides derived from interferon-alpha2b and interferon-alpha14c produced by Sendai-virus-induced human peripheral blood leukocytes

We have previously isolated and partially characterised the components of a highly purified interferon-alpha (IFN-alpha) preparation produced by Sendai-virus-induced human peripheral blood leukocytes. Nine IFN-alpha species were identified, and two of these were found to be glycosylated [Nyman, T. A., T?l?, H., Parkkinen, J. & Kalkkinen, N. (1998) Identification of nine interferon-alpha subtypes produced by Sendai-virus-induced human peripheral blood leukocytes, Biochem. J. 329, 295-302]. Here, we isolated the N-linked oligosaccharides of IFN-alpha14c and the O-linked chains of IFN-alpha2b, and the glycans were characterised by electrospray tandem mass spectrometry and by specific glycosidase digestions monitored by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The IFN-alpha14c N-glycans were shown to exhibit core-fucosylated biantennary glycans, with about 10% carrying an additional alpha1,3-linked fucose unit at the antennae. The IFN-alpha2b was shown to carry about 50% core type-1 disialyltetrasaccharides, 30% core type-1 monosialyltrisaccharides and 20% core type-2 monosialylpentasaccharides.     

The case of myoepithelioma of the parotid gland

STUDY OBJECTIVE: To determine whether the environment of a moving ambulance affects the ability of our-of-hospital care providers to auscultate breath sounds. METHODS: Out-of-hospital care providers assessed breath sounds with a previously described breath-sounds model in a quiet environment (control) and in a moving ambulance. The setting was a nonurban emergency medical services system and an interhospital transport agency based at a 600-plus-bed tertiary care center. The participants were physicians, transport nurses, and advanced life support EMS providers routinely involved in the emergency out-of-hospital treatment and transportation of the ill and injured. The accuracy with which participants identified the presence or absence of breath sounds in the two environments was compared with the use of the chi 2 test, with the alpha-value set at .05. RESULTS: The accuracy of breath-sounds assessment in the control environment was 96% (251 of 260); the sensitivity was 96% and the specificity 97%. The accuracy of breath-sounds assessment in the experimental environment was 54% (140 of 260); the sensitivity was .09% and the specificity 98%. Participants were significantly less likely to hear breath sounds in the moving ambulance than in the quiet room (P < .001). CONCLUSION: Assessment of breath sounds is hampered by the environment of a moving ambulance.     

Polymorphous low-grade adenocarcinoma of the parotid salivary gland

Polymorphous low-grade adenocarcinoma is an unusual tumor that has been described as occurring in the minor salivary glands, particularly of the palate. To our knowledge, there is only one previous report that has documented the occurrence of this type of lesion in the major salivary glands. We describe an elderly patient with polymorphous low-grade adenocarcinoma arising in the parotid gland that was originally diagnosed as carcinoma ex pleomorphic adenoma.     

Surgical pathology of parotid gland tumors

The records of 27 patients operated for parotid tumors were reviewed retrospectively. Pleomorphic adenoma was the most frequent tumor (37.1%) and required subtotal parotidectomy in all cases. Twenty percent presented permanent facial paralysis of the marginal mandibular branch. No recurrence has been observed in five years of follow-up. Warthin's tumor, found in 11.1% of patients, was removed by either superficial or subtotal parotidectomy. Parotidean cysts were observed in 7.4% and were excised by superficial parotidectomy. The malignant tumors included squamous cell carcinoma (22.2%), adenoid cystic carcinoma (14.8%), melanoma (3.7%), and renal-cell metastasis (3.7%). All were treated by total parotidectomy with conservation of the facial nerve in 67%. Twenty-five percent had postoperative facial paralysis and 33% developed Frey's syndrome. Thirty-three percent died in the next 5 years from locoregional metastases.     

Conserved N-linked oligosaccharides of the C-terminal portion of human immunodeficiency virus type 1 gp120 and viral susceptibility to neutralizing antibodies

We have constructed a mutated infectious HIV variant lacking the signals for addition of three N-linked glycans situated in the V4, C4 and V5 regions of HIV gp120. When comparing mutated virus with wildtype virus we found essentially no differences in the phenotypic characteristics of the two viruses except for the expected electrophoretic mobility shift of radioimmuno-precipitated mutated gp120, resulting from the missing N-glycans. Thus, the infectivity titer and the capacity to induce syncytia were similar for the two viruses. The sensitivity of mutant and wildtype virus to a number of neutralizing agents was determined. As expected, the mutant virus was significantly less sensitive to neutralization by Con A, with affinity for the N-glycans eliminated. We found, however, that antibodies to the V3 loop and sCD4 neutralized wild-type virus as efficiently as mutant virus, whereas 2G12, a monoclonal antibody, binding to a discontinuous neutralization epitope, and GP13, binding to the CD4-binding domain, neutralized wildtype virus better than mutant virus. Altogether the data suggest that the three conserved N-linked glycans, despite their location in immediate association with the CD4-binding domain, which is an important neutralization epitope, are not essential for virus replication in cell culture and they are not engaged in shielding neutralization epitopes of gp120 from neutralizing antibodies. However, the glycans evidently influence the three-dimensional conformation of gp120, since their presence increases the availability of the neutralization epitope of 2G12.