Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Identification of an Epstein-Barr virus-specific desoxyribonuclease gene using complementary DNA.

We have recently obtained 18 distinct cDNA clones representing different genes expressed in the early phase of EBV infection. One of them, c37, which is situated at the position 12907-122451 in the B95-8 viral genome, is shown here to code for a viral desoxyribonuclease [DNase]. Cell free translation of c37-selected messenger RNA yielded a protein of about 52 KDa which was immunoprecipitated by a high EA titer serum from nasopharyngeal carcinoma patient. This protein showed a DNase activity which was resistant to high salt concentrations (150 to 300 mM KCl) and was specifically neutralized by EA positive serum. These properties are typical of the EBV-specific DNase activity that we recently described in chemically induced EBV-transformed lymphoid cells. The same results were obtained on cell-free translation of the native RNA synthesized in vitro from pGEM-37 plasmid containing the entire c37 cDNA sequence (1.53 Kb). These data indicate that the BGLF5 open reading frame contained in c37 encodes for the EBV-specific DNase.     

Constructing verb paradigms in French: adult construals and emerging grammatical contrasts

Children acquiring French at first use just one form of each verb, where the meaning identifies only the event-type. How and when do they add additional forms, with the appropriate meanings? In this paper, we explore the information available to children from adult reformulations of these early verb-form uses, and show how adult construals contribute to children’s learning to distinguish between the homophonous infinitival (donner ‘give-Inf’) and participial (donné ‘give-PP’) forms, both /done/, of class 1 verbs. We argue that conversational exchanges, with the construals adults contribute, enable children to differentiate the meanings of homophonous forms, and so begin to construct verb paradigms.     

Amino acid composition of sperm histones in the house cricket Acheta domesticus.

Histones were isolated from late spermatids and spermatozoa of the house cricket Acheta domesticus, and the individual histone fractions were separated by electrophoresis on polyacrylamide-urea gels. The stained gels were cut so as to isolate the different histone fractions, and the amino acid compositions were determined using the technique of Houston (Houston, L.L.: Anal. Biochem. 44, 81-88 (1971). Five of the histones had amino acid compositions resembling those for the histones of calf thymus and were thus identified as fractions F1, F3, F2a2, F2b, and F2al. Another protein (SH) located exclusively in the late spermatids and spermatozoa was found to be basic and histone-like. It is a protein containing relatively high amounts of arginine (12.6%) and low amounts of lysine (7.6%), and, as a result, it has a low ratio of lysine-arginine (0.6). Other noteworthy features are its high contents of serine, glutamic acid, and glycine. It is arginine rich histone and in this regard resembles other such proteins, but it does contain unique features which distinguish it from all previously described histones.