Protease substrate specificity mapping using membrane-bound peptides

A method is described for assessing the substrate specificity of proteases by screening for proteolytic activity against large numbers of peptides. All 400 possible peptides derived from the 20 common amino acids were synthesized on small membrane disks in the arrangement FTC-spacer-amino acid P1-amino acid P'1-spacer-membrane, where FTC is a chromophoric group. The disks are incubated simultaneously with the protease, resulting in cleavage of the peptide between the P1 and P'1 amino acids, and the absorbance of the released chromophore is measured as a function of time. As demonstrated for chymotrypsin and papain, plots of the resulting data present a perspective view of the amino acid preferences on both sides of the scissile bond. This technique is fast, requires relatively little enzyme, and can be extended to the systematic screening of longer peptides, including analogs with unnatural amino acids. It has potential use for characterizing the specificity of proteases, assessing the results of site-specific mutagenesis, and searching for optimal substrates and inhibitors.     

Identification of 4(-) states in the 14C(p,n)14N reaction at 135 MeV

The tadpole larva of solitary ascidians has 40 notochord cells in its tail. Of these cells, 32 in the anterior and middle part of the tail are derived from the A-line blastomeres, while 8 in the posterior part of the tail originate from the B-line blastomeres. Previous experiments involving continuous dissociation of daughter blastomeres from the first cleavage to the 110-cell stage suggested that cellular interactions may be involved in the formation of notochord cells. In the present study, the presumptive-notochord blastomeres isolated from the 32-cell embryos did not develop features of notochord. These results suggest that cellular interactions may be required for the fate specification of notochord, that is to say, notochord formation occurs as a result of inductive interaction between blastomeres. In order to confirm the involvement of induction in the determination of notochord and to identify the inducer blastomeres, the presumptive-notochord blastomeres at the 32-cell stage were coisolated or recombined with one of the surrounding blastomeres in a series of experiments. The results suggested that, for the A-line precursors, notochord differentiation occurs as the result of an inductive influence from vegetal blastomeres that include the presumptive-endoderm blastomeres and the presumptive-notochord blastomeres themselves. It was also suggested that induction of notochord is complete by the 64-cell stage and that inductive interactions have to be initiated before the decompaction of blastomeres during the 32-cell stage. Ascidians are Urochordata and are closely related to vertebrates. In vertebrates, it is well known that inductive interactions play a crucial role in the determination of notochord. It appears, therefore, that induction of notochord is common throughout the phylum Chordata.     

Genetically engineered antibodies for diagnostic pathology

Antibody genes can be cloned, genetically manipulated, and expressed in both homologous and heterologous expression systems to produce viable antigen-binding proteins complete with natural effector functions. Manipulation of antibody genes permits the expression of fusion proteins or truncated proteins that retain antigen-binding activity. The new antibody technologies are becoming increasingly sophisticated, permitting the alteration of antigen-binding responses, the transfer of antigen specificity between antibodies, and the expression of minimal-size antigen-binding protein domains. These new molecules have been made mostly for studies on function or to provide molecules suited for in vivo diagnosis and therapy; very few have been specifically designed for, or used for, diagnostic histopathology. We describe here the adaptation of small antibody derivatives for use in immunohistochemistry. Molecules suitable for this purpose need only to possess specific antigen-binding ability and some means of detection of antigen-bound material. Detection could be by recognition of a genetically fused flag or tag epitope, by the fusion of an enzyme whose activity can be assayed, or by fusion with a protein that can interact with pre-existing histopathological reagents.     

Radioimmunoassay of regulatory peptides in the presence of acetonitrile: marked improvement of cholecystokinin assays

Radioimmunoassay has made it possible to measure the levels of many hormones. However, samples for some hormones, such as cholecystokinin (CCK), need to be purified by reverse phase chromatography before assay. Usually, samples are eluted from cartridges or HPLC columns in about 50% acetonitrile, dried on a vacuum centrifuge, and then reconstituted in buffer. Drying and reconstituting samples is time consuming and introduces additional sources of error and peptide loss. The present study investigated the effect of acetonitrile on radioimmunoassays for CCK to see if samples containing acetonitrile could be assayed directly. The non-specific binding of a radiolabeled peptide, the zero binding (B0), and the fall in the presence of 2.5 fmol unlabeled CCK were determined in the presence of various proportions of acetonitrile with 0.1% TFA. Additionally, standard curves were compared in the presence and absence of 200microl of 50% acetonitrile, (n = 5). For assays using two separate CCK antisera, increasing amounts of acetonitrile gave progressively higher zero binding and fall, thereby increasing sensitivity and antibody titer. The use of 200microl 50% acetonitrile, chosen to represent typical sample conditions, increased antiserum titers by three to four-fold, as well as increasing sensitivity considerably. For one antiserum (CCK2), the IC20 was 0.36+/-0.02 fmol CCK/tube in the presence of acetonitrile and 1.45+/-0.08 fmol/tube in its absence (P< 0.001). For the other antiserum (Dino 7), the IC20 was 0.40+/-0.02 fmol CCK/tube in the presence of acetonitrile and 0.63+/-0.01 fmol/tube in its absence (P<0.001). A similar increase in sensitivity was seen with a gastrin assay. However, no significant change in the gastrin antibody titer was evident. Assays for several other hormones were unaffected by 200 microl of 50% acetonitrile. At volumes encountered in samples following chromatography, acetonitrile did not adversely affect radioimmunoassays for a number of hormones, and the sensitivity and antibody titer of the CCK assays were improved. Measurement of CCK samples without drying and reconstitution increases assay efficiency and sensitivity.     

Effect of immunomodulators in the hu-PBL-SCID mouse model

The effects of two immunomodulators were investigated in severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID mice). Both immunomodulators, maleic anhydride divinyl ether (MVE-2) and 4-imino-1,3-diazobicyclo-(3.1.0)-hexan-2- one (imexon), have been previously studied by us in retrovirus-infected mice. To determine the effects of these compounds as they may function in humans, 24 SCID mice were each reconstituted with 20 x 10(6) ficoll-purified lymphocytes from a single donor. Five weeks after reconstitution, the mice received 16 mg/kg/day of MVE-2 intraperitoneally (i.p.) on days 0, 7, and 14 or 110 mg/kg/day of imexon i.p. daily for 14 days. Spleens were removed and splenocytes labeled with monoclonal antibodies for T- and B-cell enumeration as determined by flow cytometry 24 h after final treatment. Imexon-treated mice demonstrated a slight increase in total T cells and T cell subsets compared to control mice. T helper/T suppressor cell ratios in imexon-treated mice were brought to a normal 3:2 ratio compared to placebo-treated mice. Human immunoglobulin levels were markedly increased in imexon-treated mice. MVE-2-treated hu-PBL-SCID mice had significantly reduced numbers of total T cells compared to controls. The T-cell population results using human cells in SCID mice were similar to the effects of these immunomodulators on murine cells in immunologically competent mice.