N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.     

Host factors that influence the duration of menstrual bleeding

We assessed the association between weight, exercise, and stress and the duration of menstrual bleeding in a 1-year prospective menstrual diary study of 166 college first-year women, age 17-19 years. Low weight-for-height increased expected bleed duration by 0.39 day. Dieting to lose weight reduced bleed length by 0.43 day. Women who did no moderate or hard exercise bled about a quarter of a day longer than women at the median level of physical activity.     

Synovial sarcoma of the suboccipital region of the neck

A synovial sarcoma (SS) is an uncommon malignant soft-tissue tumor, which in spite of its name does not arise from synovial tissue. It is so named because of its histologic similarity to synovium. An SS originates from mesenchyme, not from synoviocytes and usually manifests as a biphasic tumor with both malignant-epithelial and spindle-cell components. Monophasic epithelial and spindle-cell presentations may cause a diagnostic dilemma. Diagnosis should include immunocytochemistry using cytokeratin and/or epithelial membrane antigen; vimentin further helps to eliminate any histologic confusion. These tumors are most commonly found in the extremities. When located near a joint, invasion occurs only by secondary extension. Rarely are SSs found in the neck, especially in the posterior aspect, as reported here.     

Exclusion of the neurofibromatosis 1 locus in a family with inherited café-au-lait spots

We have performed linkage analysis in a small family with autosomal dominant inheritance of multiple café-au-lait spots (CLS) in order to clarify its relationship to classical von Recklinghausen disease (NF 1). We found that an affected woman had transmitted a different haplotype for markers flanking the NF1 gene to both of her affected daughters. These results exclude an allelic mutation of the NF 1 gene on chromosome 17 as the cause for inherited café-au-lait spots in this family.     

Chiroinositol deficiency and insulin resistance. I. Urinary excretion rate of chiroinositol is directly associated with insulin resistance in spontaneously diabetic rhesus monkeys

Previously, we demonstrated that nondiabetic insulin-resistant monkeys had reduced covalent insulin activation of muscle glycogen synthase (GS) compared to normal monkeys and that covalent insulin activation of adipose tissue GS was absent in these monkeys. Covalent insulin activation of muscle and adipose tissue GS in monkeys with impaired glucose tolerance and noninsulin-dependent diabetes (NIDDM) was also absent. As in humans, monkeys with NIDDM have a lower urinary excretion rate of chiroinositol (CI), a component of a putative mediator of insulin action, compared to normal monkeys. To determine whether the urinary excretion rate of CI was related to insulin resistance, which develops naturally in many obese rhesus monkeys, we examined the relationships between 24-h urinary CI excretion rate and 1) whole body insulin-mediated glucose disposal rates (M) and insulin-mediated changes in 2) the skeletal muscle GS activity ratio (sm delta GSAR), 3) the skeletal muscle glycogen phosphorylase activity ratio, and 4) the adipose tissue GS activity ratio (at delta GSAR) in 27 monkeys ranging from normal (n = 12) to insulin resistant (n = 8) to overtly diabetic (n = 7). The urinary CI excretion rate was significantly correlated with M (r = 0.47; P < 0.02), sm delta GSAR (r = 0.38; P < 0.05), skeletal muscle glycogen phosphorylase activity ratio (r = -0.49; P < 0.01), and at delta GSAR (r = 0.46; P < 0.02). The urinary CI excretion rate was also correlated with glucose tolerance (r = 0.39; P < 0.05). There was a wide range of urinary CI excretion rates (0.42-5.17 mumol/day) in monkeys with normal fasting plasma glucose concentrations. However, of the 7 diabetic monkeys, 6 had a urinary CI excretion rate below 2.0 mumol/day, and in the subgroup of 16 monkeys with a urinary CI excretion rate less than 2.0 mumol/day, the associations of urinary CI with M rate (r = 0.65; P < 0.005), glucose tolerance (r = 0.63; P < 0.01), and sm delta GSAR (r = 0.73; P < 0.001) increased in strength and significance. We propose that the urinary CI excretion rate may be 1) a biochemical indicator of both in vivo and in vitro insulin resistance and 2) a noninvasive diagnostic tool with potential for the identification of those individuals at risk for NIDDM and other related diseases with insulin resistance.     

A comparison of two murine monoclonal antibodies humanized by CDR-grafting and variable domain resurfacing

The variable domain resurfacing and CDR-grafting approaches to antibody humanization were compared directly on the two murine monoclonal antibodies N901 (anti-CD56) and anti-B4 (anti-CD19). Resurfacing replaces the set of surface residues of a rodent variable region with a human set of surface residues. The method of CDR-grafting conceptually consists of transferring the CDRs from a rodent antibody onto the Fv framework of a human antibody. Computer-aided molecular modeling was used to design the initial CDR-grafted and resurfaced versions of these two antibodies. The initial versions of resurfaced N901 and resurfaced anti-B4 maintained the full binding affinity of the original murine parent antibodies and further refinements to these versions described herein generated five new resurfaced antibodies that contain fewer murine residues at surface positions, four of which also have the full parental binding affinity. A mutational study of three surface positions within 5 A of the CDRs of resurfaced anti-B4 revealed a remarkable ability of the resurfaced antibodies to maintain binding affinity despite dramatic changes of charges near their antigen recognition surfaces, suggesting that the resurfacing approach can be used with a high degree of confidence to design humanized antibodies that maintain the full parental binding affinity. By comparison CDR-grafted anti-B4 antibodies with parental affinity were produced only after seventeen versions were attempted using two different strategies for selecting the human acceptor frameworks. For both the CDR-grafted anti-B4 and N901 antibodies, full restoration of antigen binding affinity was achieved when the most identical human acceptor frameworks were selected. The CDR-grafted anti-B4 antibodies that maintained high affinity binding for CD19 had more murine residues at surface positions than any of the three versions of the resurfaced anti-B4 antibody. This observation suggests that the resurfacing approach can be used to produce humanized antibodies with reduced antigenic potential relative to their corresponding CDR-grafted versions.     

Monoclonal antibodies specific to the acute lymphoblastic leukemia t(1;19)-associated E2A/pbx1 chimeric protein: characterization and diagnostic utility

Nonrandom chromosomal abnormalities are found in most human malignancies, particularly leukemias and lymphomas. A characteristic t(1;19) (q23;p13.3) chromosomal translocation is detected in 5% of childhood acute lymphoblastic leukemia (ALL) cases. This translocation results in the formation of a fusion gene, which leads to the expression of an oncogenic E2A/pbx1 protein. Breakpoints in the E2A gene almost invariably occur within a single intron, and the identical portion of PBX1 is joined consistently to exon 13 of E2A in fusion mRNA. In this article, we report the development of monoclonal antibodies against E2A/pbx1 fusion protein using a specific peptide that corresponds to the junction region of the protein. The obtained antibodies recognize specifically the chimeric E2A/pbx1 fusion protein and lack cross-reactivities with E2A and pbx1. Immunohistochemical staining and flow cytometric studies show that these antibodies can distinguish t(1;19)-positive from t(1;19)-negative leukemic cells. These results indicate that the obtained E2A/pbx1-specific monoclonal antibodies might prove to be valuable diagnostic reagents and important tools for elucidating the mechanisms involved in oncogenesis and progression of t(1;19)-positive childhood ALL.