The monosaccharide content of a glycoprotein is often determined by acid hydrolysis at elevated temperature and subsequent high pH chromatography of the released, underivatized monosaccharides on pellicular anion-exchange resin (HPAE) using pulsed amperometric detection (PAD). We have found that for glycoproteins with low levels of glycosylation, monosaccharide quantitation can be compromised by amino acids fouling the working electrode surface. Specifically, lysine elutes on the CarboPac PA1 column just prior to galactosamine, whereas remaining amino acids and most peptides elute after the monosaccharides and do not interfere with monosaccharide quantification.
View Article and Find Full Text PDFThrombocytopenia may be the presenting finding for both Wiskott-Aldrich syndrome and Fanconi anemia. We examined a sibship of four boys who had features of both of these hematologic disorders. Peripheral blood lymphocytes from three of the boys demonstrated DNA instability when cultured with diepoxybutane, confirming the diagnosis of Fanconi anemia in these patients.
View Article and Find Full Text PDFProtein Expr Purif
February 1996
Glycoproteins are microheterogeneous with respect to their attached oligosaccharides. When these oligosaccharides contain sialic acid, the oligosaccharide microheterogeneity will impart charge heterogeneity to the glycoprotein. We found that two commercial preparations of human serum transferrin (HST), a sialylated glycoprotein, have very different chromatographic profiles when the samples are separated by pellicular anion-exchange chromatography.
View Article and Find Full Text PDFWe have examined whether the two cysteine residues (Cys30 and Cys34) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-PAGE. Both Cys30 and Cys34 were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor.
View Article and Find Full Text PDFLamp1 is a type I transmembrane glycoprotein that is localized primarily in lysosomes and late endosomes. Newly synthesized molecules are mostly transported from the trans-Golgi network directly to endosomes and then to lysosomes. A minor pathway involves transport via the plasma membrane.
View Article and Find Full Text PDFMutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed.
View Article and Find Full Text PDFWe have reported that in irradiated, long-term surviving RFM strain of mice there is enhanced kinetics of tumor development upon challenge with RFM lymphoma cells. We reported that we cloned splenic oncofetal (OFA)-specific, noncytotoxic CD8+ T cells from such mice. These noncytotoxic CD8+ T cell clones secrete a factor upon Ag stimulation that inhibits the ability of OFA-specific RFM cytotoxic T (TC) cell clones from killing 5T RFM lymphoma cells in vitro.
View Article and Find Full Text PDFObjective: This study investigates the determinants of primary care office visit rates.
Data Sources: Blue Cross and Blue Shield of Iowa subscriber information was sorted by residence into geographic health service areas. Cost-sharing information was also obtained from Blue Cross.
The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) is a type 1 transmembrane protein that cycles between the trans-Golgi network, endosomes, and the plasma membrane. When the terminal 40 residues were deleted from the 67-amino acid cytoplasmic tail of the CD-MPR, the half-life of the receptor was drastically decreased and the mutant receptor was recovered in lysosomes. Analysis of additional cytoplasmic tail truncation mutants and alanine-scanning mutants implicated amino acids 34-39 as being critical for avoidance of lysosomal degradation.
View Article and Find Full Text PDFClin Immunol Immunopathol
September 1995
p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane.
View Article and Find Full Text PDFThe following two tri-sialylated triantennary oligosaccharides, which differ only in the linkage of the Neu5Ac to the uppermost branch were, individually, partially desialylated to produce all possible di- and mono-sialylated isomers. [formula: see text] A tetra-sialylated triantennary isomer, which contained an alpha (2-->6)-linked Neu5Ac to the GlcNAc on branch III, was also converted to all possible trisialylated isomers by mild acid hydrolysis as previously described (Roher et al., Anal.
View Article and Find Full Text PDFPalmitoylation of p63, a type II membrane protein localized in the endoplasmic reticulum, is induced in a reversible manner by the drug brefeldin A. To study the requirements for palmitoylation, mutant forms of p63 were expressed in COS cells and analyzed by metabolic labeling with [3H]palmitate, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis. By investigating deletion and point mutations, Cys100 in the 106-amino acid cytoplasmic tail of p63 has been identified as the site of acylation.
View Article and Find Full Text PDFHealth Serv Manage Res
May 1995
Rural hospitals have been threatened by declining revenues. Control over costs will be necessary to help these hospitals survive. Investigation of the determinants of hospital costs in Iowa reveals that costs are primarily caused by environmental factors, rather than variables over which managers have control.
View Article and Find Full Text PDFFractionated sublethal x-irradiation induces thymic lymphomas in up to 60% of RFM mice by 6 mo after irradiation, but no thymomas arise after 6 mo. All radiation-induced tumors expressed oncofetal Ag (OFA) and thymic OFA expression significantly preceded detectable thymomas. To determine whether lymphoma-free, radiation survivor mice had anti-OFA T cell immunity, we analyzed their clonable 5T lymphoma-reactive T lymphocytes and determined the resistance of such mice to challenge with RFM lymphoma cells.
View Article and Find Full Text PDFMonitoring newly emerging "accountable health plans" is a public health responsibility related to Certificate-of-Need regulation. AHPs should use locally-based primary care providers, assure access to high quality affordable care to rural communities, contract with essential community providers, be governed by local boards of health, and report information necessary to evaluate their performance. Performance should be measured epidemiologically, using population-based rates such as infant mortality, incidence of late-stage breast and cervical cancer, and avoidable hospitalizations.
View Article and Find Full Text PDFBTK, the gene that is defective in X-linked agammaglobulinemia, encodes a cytoplasmic tyrosine kinase that is critical for B-cell proliferation, or survival. To identify regulatory elements that control the expression of BTK we evaluated the methylation pattern of this gene in cell lines and in freshly isolated cells. An Hpa II site that was specifically demethylated in mature B cells but not in pre-B cells, T cells, neutrophils, or nonhematopoietic cells was identified in the tenth intron of BTK.
View Article and Find Full Text PDFClin Perform Qual Health Care
May 1995
The effectiveness of the local medical system may be evaluated using hospital discharge rates. The method is illustrated using the case of preventable pediatric admissions. Results of the analysis support the validity of admission rates for bronchitis and asthma, pneumonia, gastroenteritis, and otitis media as indicators of the primary care delivery system.
View Article and Find Full Text PDFMutations in the gene for Bruton's tyrosine kinase (Btk) are responsible for X-linked agammaglobulinemia (XLA). Thus far, mutations in this gene have been identified based on alterations in Southern or Northern blot analysis or cDNA sequence. To permit detection of mutations in genomic DNA, we designed PCR primers to flank each of the 19 exons of Btk with splice sites.
View Article and Find Full Text PDFThe type II membrane protein p63 is a resident protein of a membrane network interposed between rough ER and Golgi apparatus. To study the retention of p63, mutant forms were expressed in COS cells and the intracellular distribution determined by immunofluorescence microscopy. Investigation of chimeric constructs between p63 and the plasma membrane protein dipeptidylpeptidase IV showed that protein sequences from all three domains of the p63 protein are required to achieve complete intracellular retention.
View Article and Find Full Text PDFThe glycoprotein bovine fetuin was treated with trypsin and the Asn-81 tryptic glycopeptide was purified (90% pure by Edman sequencing) by reversed-phase chromatography (RP-HPLC). The Asn-81 glycopeptide, which eluted as a single peak by RP-HPLC, was separable into five peaks on the NucleoPac PA100 column, a pellicular anion-exchange column. Each of the five Asn-81 glycopeptide peaks was shown to contain N-linked oligosaccharides by treatment of each peak with peptide N4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F (PNGase F) and subsequent oligosaccharide analysis by high-pH anion-exchange chromatography with pulsed amperometric detection.
View Article and Find Full Text PDFThe identification of a cytoplasmic tyrosine kinase, Btk, as the defective protein in human XLA and xid in the mouse, supports the hypothesis that both disorders are due to defects in B-cell activation or differentiation. Phenotypic analysis of B-lineage cells and studies on X-chromosome inactivation patterns in both mice and human patients suggest that mutations in Bth do not affect entry of stem cells into the B-lineage pathway but they do inhibit progression at multiple steps along that pathway. Although the exact function of Btk in signal transduction is not yet known, it is probable that studies which correlate specific mutations in different patients with alterations in Btk function will provide clues about critical sites in the molecule.
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