Detection of Bacillus anthracis in animal tissues using InBios active anthrax detect rapid test lateral flow immunoassay.

The Active Anthrax Detect (AAD) Rapid Test lateral flow immunoassay is a point-of-care assay that was under investigational use for detecting Bacillus anthracis capsular polypeptide (polyglutamic acid) in human blood, serum and plasma. Small sample volumes, rapid results and no refrigeration required allow for easy use in either the field or laboratory. Although the test was developed for use in suspect cases of human inhalation anthrax, its features also make it a potentially powerful tool for testing suspect animal cases. We tested animal tissue samples that were confirmed or ruled out for B. anthracis. The AAD Rapid Tests were also deployed in the field, testing animal carcasses during an anthrax outbreak in hippopotami (Hippopotamus amphibius) and Cape buffalo (Syncerus caffer) in Namibia. Evaluation of all samples showed a specificity of 82% and sensitivity of 98%. However, when the assay was used on specimens from only fresh carcasses (dead for <24 h), the specificity increased to 96%. The AAD Rapid Test is a rapid and simple screening assay, but confirmatory testing needs to be done, especially when the age of the sample (days animal has been deceased) is unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: In countries where anthrax is endemic, many human outbreaks are often caused by epizootics. Earlier detection of infected animals may allow for identification of exposed people, early implementation of prevention and control methods, and ultimately lessen the number of people and animals affected. Detection of Bacillus anthracis in animal tissues using a simple, rapid and field-deployable method would allow for faster outbreak response. We evaluated a simple sample collection and processing method for use with the Active Anthrax Detect Rapid Test lateral flow immunoassay to screen dead animals for anthrax.

Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

BACKGROUND: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. METHODS: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case-control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. RESULTS: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. CONCLUSION: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

Collagen fiber regulation in human pediatric aortic valve development and disease.

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

MUC4 expression is regulated by cystic fibrosis transmembrane conductance regulator in pancreatic adenocarcinoma cells via transcriptional and post-translational mechanisms.

MUC4 mucin is a high molecular weight transmembrane glycoprotein that plays important roles in tumour biology. It is aberrantly expressed in pancreatic adenocarcinoma, while not being detectable in the normal pancreas. Previous studies have demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is defective in CF, is implicated in multiple cellular functions, including gene regulation. In the present study, using a CFTR-defective pancreatic cancer cell line and its derived subline expressing functional CFTR, we report that MUC4 expression is negatively regulated by CFTR. Short-interfering RNA (siRNA)-mediated silencing of CFTR also leads to an increased expression of MUC4. Additionally, our results suggest that CFTR-mediated regulation of MUC4 is cell density-dependent and is achieved by transcriptional and posttranslational mechanisms. Moreover, in a panel of pancreatic cancer cell lines and normal pancreas, we observed that CFTR was downregulated in pancreatic cancer cells and negatively correlated with MUC4 in most cases. An aberrant expression of MUC4 was also detected in the CF pancreas. Downregulation of CFTR in pancreatic adenocarcinoma and its inverse association with the tumour-linked mucin, MUC4, indicate novel function(s) of CFTR in pancreatic tumour biology and suggest the implication of new signalling pathway(s) in MUC4 regulation.

Early diagnosis of pancreatic cancer: neutrophil gelatinase-associated lipocalin as a marker of pancreatic intraepithelial neoplasia.

Pancreatic cancer is a highly lethal malignancy with a dismal 5-year survival of less than 5%. The scarcity of early biomarkers has considerably hindered our ability to launch preventive measures for this malignancy in a timely manner. Neutrophil gelatinase-associated lipocalin (NGAL), a 24-kDa glycoprotein, was reported to be upregulated nearly 27-fold in pancreatic cancer cells compared to normal ductal cells in a microarray analysis. Given the need for biomarkers in the early diagnosis of pancreatic cancer, we investigated the expression of NGAL in tissues with the objective of examining if NGAL immunostaining could be used to identify foci of pancreatic intraepithelial neoplasia, premalignant lesions preceding invasive cancer. To examine a possible correlation between NGAL expression and the degree of differentiation, we also analysed NGAL levels in pancreatic cancer cell lines with varying grades of differentiation. Although NGAL expression was strongly upregulated in pancreatic cancer, and moderately in pancreatitis, only a weak expression could be detected in the healthy pancreas. The average composite score for adenocarcinoma (4.26+/-2.44) was significantly higher than that for the normal pancreas (1.0) or pancreatitis (1.0) (P<0.0001). Further, although both well- and moderately differentiated pancreatic cancer were positive for NGAL, poorly differentiated adenocarcinoma was uniformly negative. Importantly, NGAL expression was detected as early as the PanIN-1 stage, suggesting that it could be a marker of the earliest premalignant changes in the pancreas. Further, we examined NGAL levels in serum samples. Serum NGAL levels were above the cutoff for healthy individuals in 94% of pancreatic cancer and 62.5% each of acute and chronic pancreatitis samples. However, the difference between NGAL levels in pancreatitis and pancreatic cancer was not significant. A ROC curve analysis revealed that ELISA for NGAL is fairly accurate in distinguishing pancreatic cancer from non-cancer cases (area under curve=0.75). In conclusion, NGAL is highly expressed in early dysplastic lesions in the pancreas, suggesting a possible role as an early diagnostic marker for pancreatic cancer. Further, serum NGAL measurement could be investigated as a possible biomarker in pancreatitis and pancreatic adenocarcinoma.

The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis.

The 19q13 amplicon in pancreatic cancer cells contains a novel pancreatic differentiation 2 (PD2) gene (accession number AJ401156), which was identified by differential screening analysis. PD2 is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1). In yeast, Paf1 is part of the transcription machinery, acting as a docking protein in between the complexes Rad6-Bre1, COMPASS-Dot1p, and the phosphorylated carboxyl terminal domain of the RNA polymerase II. As such, Paf1 is directly involved in transcription elongation via histone H2B ubiquitination and histone H3 methylation. The PD2 sequence is highly conserved from Drosophila to humans with up to 98% identity between rodent and human, suggesting the functional importance of PD2/hPaf1 to maintain cellular homeostasis. PD2 is a modular protein composed of RNA recognition motif, DEAD-boxes, an aspartic/serine (DS)-domain, a regulator of the chromosome condensation domain and myc-type helix-loop-helix domains. Our results further showed that PD2 is a nuclear 80 kDa protein, which interacts with RNA polymerase II. In addition, we have demonstrated that the overexpression of PD2 in the NIH 3T3 cells result in enhanced growth rates in vitro and tumor formation in vivo. Altogether, this paper presents strong evidence that the overexpression of PD2/hPaf1 is involved in cancer development.

The rate of synthesis of glycosaminoglycans and collagen by fibroblasts cultured from adult human liver biopsies.

Adult human liver biopsies were cultured from normal, alcoholic hepatitis, chronic active hepatitis, fibrosis plus alcoholic hepatitis (active cirrhosis), inactive cirrhosis, and drug hepatitis. The synthesis of collagen was estimated in cultures from 58 livers by measuring the conversion of [(14)C]proline to the [(14)C]hydroxyproline of collagen; that of glycosaminoglycans in cultures from 57 livers by the incorporation of [(3)H]acetate and (35)SO(4) into glycosaminoglycans (GAG). The synthesis of procollagen was increased only in cultures from alcoholic hepatitis, both in the pulse medium (P < 0.05) and in the chase medium (P < 0.02). The synthesis of insoluble collagen was increased in cultures from chronic (active) hepatitis (P < 0.01), fibrosis plus alcoholic hepatitis (active cirrhosis) (P < 0.001), and inactive cirrhosis (P < 0.05). Essentially all radioactive GAG was soluble in culture media. The predominant GAG were chondroitin-4 or -6-SO(4). The synthesis of GAG was increased only in cultures from fibrosis plus alcoholic hepatitis (active cirrhosis) both in the pulse medium (P < 0.01) and chase medium (P < 0.001). The data indicate that in the absence of immuno-competent cells or their secretory products, tissue cultures from livers showing biopsy evidence of active fibrosis in vivo may demonstrate increased synthesis of collagen and GAG in vitro. Increased (soluble) procollagen synthesis in cultures from alcoholic hepatitis was not associated with histologically demonstrable overt hepatic fibrosis in vivo, nor was it associated with increased GAG synthesis in vitro. No significant difference was demonstrable in collagen or GAG synthesis in paired cultures which contained either 300 mg/dl ethanol or 3.75 mg/dl methylprednisolone compared to their respective controls.

Helper factor(s) generated by tumor-immune rats for lymphoproliferative responses to syngeneic tumor cells.

New Zealand Black ( NBR ) rats that are innately immune to challenge with a syngeneic 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced fibrosarcoma have spleen cells that produce helper effects for in vitro lymphoproliferative responses in the presence of individual MCA-induced fibrosarcoma cells. Spleen cells from MCA-induced fibrosarcoma progressor rats (which lack innate tumor immunity) do not produce demonstrable helper activity. Supernatants from 48-hour cocultures of spleen cells from tumor-immune (TI) rats and syngeneic MCA-induced fibrosarcoma cells replaced the spleen cell helper activity. Comparable spleen cell supernatants from tumor progressor rats or unchallenged rats (controls) as well as supernatants from MCA-induced fibrosarcoma cells cultured alone did not produce any helper factor activity. Supernatants from TI rat spleen cells following inoculation with MCA-induced fibrosarcoma cells did not affect lymphoproliferative responses of NBR spleen cells induced by concanavalin A or alloantigens. The supernatants also did not contain detectable interleukin 2 activity as determined with the use of the thymocyte costimulator assay. These data indicate that the production of soluble helper factors by TI rat spleen cells may be involved in the augmentation of a protective host antitumor response.

Differences in spleen suppressor cell content in rats with progressing and regressing tumors.

Young New Zealand Black rats (less than 35 wk old) developed progressively growing tumors when given injections of cells from a syngeneic 3-methylcholanthrene (MCA)-induced fibrosarcoma. Spontaneous tumor regression occurred in animals more than 10 months of age. Compared to the responses of spleen cells from age-matched controls, spleen cells from rats in which tumor progression was present progressor rats) showed decreased proliferative responses to concanavalin (Con A), phytohemagglutinin (PHA), and syngeneic MCA tumor cells. Spleen cells from rats in which the tumors had regressed (regressor rats), however, produced significant increases in proliferative responses when compared with the responses of spleen cells from age-matched controls and from progressor rats. Cell-mixing experiments implicated the presence of two spleen suppressor cell populations in progressor rats, one of which was not present in regressor rats. The unique suppressor cell found in progressor rats appeared to be specific for tumor cell-induced proliferative responses. Spleen cells from both progressor and regressor rats produced similar suppressive effects in the PHA and Con A responses of normal cells.

Polypeptide core of a human pancreatic tumor mucin antigen.

This work describes the molecular properties of the polypeptide core of a human pancreatic mucin antigen isolated from a human pancreatic adenocarcinoma cell line, HPAF. Pancreatic tumor mucin was isolated by a combination of molecular sieve chromatography and CsCl/4 M guanidine-HCl density gradient ultracentrifugation. Trifluoromethane sulfonic acid was used to remove carbohydrate units from purified mucin molecules. A rabbit monospecific polyclonal antibody was generated against pancreatic apomucin and reacted with a Mr greater than 200,000 species. The antibody binding data indicated that the rabbit antiserum raised against pancreatic apomucin cross-reacted with a breast mucin synthetic peptide. Northern blot and immunodot blot analyses of various cell line extracts revealed that a tandem repeat sequence and a similar mRNA were detected in both pancreatic and breast mucin-producing cell lines. These results suggest that pancreatic apomucin and breast apomucin share some similarity in tandem repeated nucleic acid and protein sequences.

A novel ribonucleoprotein complex defined by monoclonal antibodies to NIH 3T3 cells transfected with human pancreatic adenocarcinoma DNA.

Three monoclonal antibodies elicited to NIH 3T3 cells transfected with DNA from a human pancreatic adenocarcinoma cell line recognized a novel ribonucleoprotein complex. Minimally, this ribonucleoprotein complex contained a Mr 240,000 protein (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and two RNA species with apparent sizes of 1.5 and 3.0 kilobases (by formaldehyde agarose gel electrophoresis). In addition to a cytoplasmic and nuclear subcellular localization, the RNA antigen was secreted from human tumor cell lines and NIH 3T3 cells transfected with pancreatic tumor DNA (inhibitable by monensin) and was apparently not a viral or Mycoplasma contaminant. The ribonucleoprotein antigen was detected in some normal tissues by immunoperoxidase but was not found in or secreted from in vitro cultured normal human fibroblasts, nontransfected or spontaneously transformed NIH 3T3 cells, or normal peripheral blood leukocytes.

Antigens expressed on NIH 3T3 cells following transformation with DNA from human pancreatic tumor.

This report documents that the pancreatic adenocarcinoma cell line, HPAF, contains oncogene activity detected by transformation of NIH 3T3 cells through transfection with HPAF DNA. The HPAF transfected NIH 3T3 cells do not contain oncogenes homologous with c-H-ras, c-K-ras, c-N-ras, v-fms, c-myb, c-sis, v-fgr, c-mos, c-myc, c-fos, v-fes, v-src, v-erb A, v-erb B, c-N-myc, v-raf, or v-abl, other than the endogenous mouse genes. The transfectants do express proteins detected by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were not found in nontransfected NIH 3T3 cells. Monoclonal antibodies raised against the transfectants recognize proteins not found in untransfected NIH 3T3 cells that are antigenically identical to proteins found in the HPAF cells. These antigens are also detected on six other human pancreatic adenocarcinoma cell lines but show a much more restricted distribution on lymphoblastoid, melanoma, prostatic carcinoma, and normal skin fibroblast cell lines.

Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model.

The human epithelial mucin, MUC1, is a large transmembrane glycoprotein that is expressed on most simple epithelia. It is overexpressed and aberrantly glycosylated on many human epithelial tumors, including more than 90% of human breast cancers. MUC1 is of interest as an immunotherapy target because patients with breast, ovarian, and pancreatic cancers have T lymphocytes in their tumor-draining lymph nodes that can be induced to recognize and lyse MUC1-expressing tumor cells. We have produced a transgenic mouse model that expresses the human MUC1 molecule on an inbred C57Bl/6 background to investigate the effect of endogenous expression of MUC1 on the ability of mice to generate antitumor immunity to MUC1-expressing tumors. Transgenic mice expressed the human transgene in a pattern and level consistent with that observed in humans. Transgenic mice were tolerant to stimulation by MUC1 as evidenced by the ability of MUC1-expressing tumor cells to grow in these mice, whereas MUC1-expressing cells were eliminated from wild-type mice. Moreover, transgenic mice immunized with MUC1 peptides failed to exhibit immunoglobulin class switching to the IgG subtypes. These data suggest that endogenous expression of MUC1 protein by MUC1 transgenic mice induces T-cell tolerance to stimulation by MUC1. The transgenic mice will provide a useful model to investigate the mechanisms that regulate immunological tolerance to tumor antigens and will facilitate the investigation of anti-MUC1 immunotherapy formulations.

Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers.

The invasive growth of malignant cells induces an admixture of host reactions including desmoplasia, angiogenesis, and immune reactions Pancreatic cancer has a prominent and characteristic host reaction at the site of primary invasion. To obtain new insights into the process of tumor invasion, we studied global patterns of gene expression using serial analysis of gene expression in pancreatic cancer, with extension to other tumor types. Here we report a cluster of invasion-specific genes in pancreatic and other cancers. This cluster contains genes that derive from distinct components of the host reaction, including some that may be useful as diagnostic markers and therapeutic targets.

P-selectin expression in a metastatic pancreatic tumor cell line (SUIT-2).

The human pancreatic tumor cell line SUIT-2 was derived from a metastatic lesion in the liver of a patient with pancreatic adenocarcinoma. SUIT-2 and clonal cell lines derived from it show spontaneous metastasis to lung and regional lymph nodes from s.c. nude mouse xenografts and were found to express P-selectin mRNA and protein. Surface expression of P-selectin protein was increased by exposure of the pancreatic tumor cells to thrombin, oxygen radicals, and trypsin, suggesting that common cellular mechanisms for regulating P-selectin surface expression exist among platelets, endothelial cells, and these pancreatic tumor cells. The finding that P-selectin is expressed by metastatic pancreatic tumor cells demonstrates that the range of cell types that express these adhesion molecules is broader than believed previously.

N-acetylgalactosamine glycosylation of MUC1 tandem repeat peptides by pancreatic tumor cell extracts.

Synthetic peptides corresponding to the human mucin MUC1 tandem repeat domain (20 residues) were glycosylated in vitro by using UDP-N-[3H]acetyl-D-galactosamine (GalNAc) and lysates of pancreatic tumor cell lines. Results obtained with peptides of different lengths (from one to five repeats) suggest that increasing the number of tandem repeats has neither a positive nor a negative effect on the density of glycosylation along the MUC1 tandem repeat protein backbone. Purified glycopeptides were sequenced on a gas-phase sequencer, and glycosylated positions were determined by measuring the incorporated radioactivity in fractions collected following each round of Edman degradation. The results showed that two of three threonine residues on the MUC1 tandem repeat peptides were glycosylated by pancreatic tumor cell lysates at the following positons: GVTSAPDTRPAPGSTAPPAH (underlined T indicates position of GalNAc attachment). None of the serine residues were glycosylated. Determination of the mass of the glycopeptides by mass spectrometry confirmed that a maximum of two molecules of GalNAc were covalently linked to each 20-residue repeat unit in the peptides. The data presented here show that acceptor substrate specificity of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase detected in lysates of pancreatic and breast tumor cell lines is identical and is limited to some but not all threonines in the MUC1 tandem repeat peptide sequence. The influence of primary amino acid sequence on acceptor substrate activity was evaluated by using several peptides that contain single or double amino acid substitutions (relative to the native human MUC1 sequence). These included substitutions in the residues that were glycosylated and substitutions of the surrounding primary amino acid sequence. The results of these studies suggest that primary amino acid sequence, length, and relative position of the residue to be glycosylated dramatically affect the ability of peptides to serve as acceptor substrates for the UDP-GalNAc:polypeptide N-acetylgalatosaminyltransferase.

Expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases in adenocarcinoma cell lines.

The levels of mRNA expression of three UDP-N-acetyl-alpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyltransferases (GalNAc-transferases) were quantified for human adenocarcinoma cell lines from pancreas, colon, stomach, and breast. Two of the GalNAc-transferases, GalNAc-T1 and GalNAc-T2, were expressed constitutively and at low levels in most or all cell lines examined. A third GalNAc-transferase, GalNAc-T3, was differentially expressed. Well-differentiated adenocarcinoma cell lines expressed high levels and moderately differentiated cell lines expressed lower levels of GalNAc-T3. Cell lines classified as poorly differentiated failed to express GalNAc-T3 mRNA at levels that could be detected by Northern blot analysis. Differential expression of the GalNAc-T3 protein was confirmed in these cell lines by Western blotting. We propose that glycosylation in tumor cell lines may be regulated in part by differential expression of GalNAc-transferases, and we suggest that GalNAc-T3 gene expression may be a molecular indicator of differentiated adenocarcinoma.

Mucin (MUC) gene expression in human pancreatic adenocarcinoma and chronic pancreatitis: a potential role of MUC4 as a tumor marker of diagnostic significance.

PURPOSE: Mucins are important biomolecules that frequently display an altered expression under pathological conditions. In a search for a unique and reliable marker(s) specific for pancreatic adenocarcinoma, we investigated the expression of different MUC genes in pancreatic tumors and tumor cell lines, in chronic pancreatitis, and in the normal pancreas. EXPERIMENTAL DESIGN: Total RNA from 16 pancreatic tumors, 10 chronic pancreatitis tissues, 7 normal pancreas tissues, and 15 pancreatic tumor cell lines were analyzed by reverse transcription-PCR with primers specific for MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7 genes and by RNA slot blot analyses. RESULTS: Our results revealed that of all of the mucins examined, only MUC4 displayed a differential expression that was specific for pancreatic adenocarcinoma. Indeed, a substantial number of tumor tissue samples (12 of 16) and tumor cell lines (11 of 15) expressed MUC4 mRNA, whereas samples from chronic pancreatitis (0 of 10) and the normal pancreas (0 of 7) tissues failed to exhibit any detectable level of this mucin. In contrast, no significant alteration was observed in the expression of the other mucins relative to that in the normal pancreas samples. CONCLUSIONS: Overall, this work demonstrates that pancreatic mucin MUC4 is a tumor-associated mucin. Furthermore, the present study introduces a novel avenue to discriminate between pancreatic adenocarcinoma and pancreatitis. Future investigations of the role played by MUC4 in pancreatic adenocarcinoma may prove to be useful in the formulation of strategies for the diagnosis and therapeutic treatment of this malignancy.

Enzymatic and chemical probing of an S1 nuclease-sensitive site upstream from the human CFTR gene.

Similar purine/pyrimidine mirror repeat (PMR) DNA sequences have been identified in the 5'-flanking regions of the human cystic fibrosis transmembrane conductance regulator (hCFTR) and mucin (hMUC1) genes, and supercoiled (but not linearized) plasmids containing these promoter regions were previously shown to be sensitive to digestion by S1 nuclease. The PMR element derived from the hCFTR promoter region is now sub-cloned and characterized at nucleotide resolution with respect to its reactivity toward nucleases S1 and P1, and toward the chemical probes dimethyl sulfate, chloroacetaldehyde, diethylpyrocarbonate and osmium tetroxide. These probes confirm the presence, at pH 4.5 (but not at pH 7.1), of a non-B-DNA structure. This non-B-DNA structure is distinct from H-DNA, because enzymatic and chemical probing detect single-stranded character in the absence of a stable intramolecular triple helix or extruded purine strand.

A novel human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates.

A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase gene family, designated GalNAc-T7, was cloned and expressed. GalNAc-T7 exhibited different properties compared to other characterized members of this gene family, in showing apparent exclusive specificity for partially GalNAc-glycosylated acceptor substrates. GalNAc-T7 showed no activity with a large panel of non-glycosylated peptides, but was selectively activated by partial GalNAc glycosylation of peptide substrates derived from the tandem repeats of human MUC2 and rat submaxillary gland mucin. The function of GalNAc-T7 is suggested to be as a follow-up enzyme in the initiation step of O-glycosylation.