Mitogen Stimulation of Lymphocytes in Pigs with Hereditary Vitamin C Deficiency

Recently an inherited vitamin G deficiency in the pigs presumably based on an autosomal recessive gene was decribed* Homozygotes are in contrast to heterozygotes and normal pigs unable to synthesize ascorbic acid. In an experiment comprising 3 littermate pigs, 2 homozygous and 1 heterozygous for the vitamin C deficiency gene, the influence of ascorbic acid depletion, and repletion on mitogen stimulation of peripheral blood lymphocytes was studied. Ascorbic acid depletion of the vitamin C dependent pigs resulted in a rapid decline in plasma ascorbic acid. Response of lymphocytes to stimular tion with Concanavalin A (Con A) and phytohemagglutinin M (PHA) decreased more slowly reaching a minimum, which coincidedi with the occurrence of the first clinical symptoms of scurvy. Following resupplementation with vitamin C the plasma content of ascorbic acid rapidly returned to normal, while the lymphocyte response to Con A and PHA stimulation only gradually approached the initial values. The repletion with ascorbic acid caused a transitory increase in the response to pokeweed mitogen (PWM) stimulation. The significance of these findings in relation to the cellular immune system in normal pigs is discussed.     

Mechanisms of serotonin-induced lymphocyte proliferation inhibition

When human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence of serotonin, inhibition of [3H]thymidine incorporation occurred, the most marked inhibition occurring at high (10(-3)M) serotonin concentrations. This effect could not be reversed by the addition of Interleukin 2 (IL-2)-containing supernatants. Cytofluorometric analysis showed that virtually all of the cells remained in the G0 phase (unactivated) at 24 hr while some of the cells entered the G1a and G1b phases of the cell cycle by 42 hr. The cellular production of IL-2 was not affected by serotonin, as supernatants of treated cultures contained essentially the same IL-2 titers as did control cultures. Serotonin seemed to primarily affect cell activation and had little or no effect on proliferating cells. This was further confirmed by the lack of effects of serotonin on a variety of established proliferating lymphocyte, macrophage, and fibroblast cell lines. By contrast, dose-dependent inhibition of IL-2-dependent CTLL cells occurred. Serotonin was not toxic even at 10(-3) M concentrations. A marked decrease in IL-2 receptors and a change in their distribution on responder cells was seen when treated cultures were examined with the anti-Tac monoclonal antibody. At 24 hr this effect was contrastingly not seen for the OKT-8 marker, although a slight decrease in OKT-4-positive cells was seen. Serotonin thus produced an inhibition of lectin-stimulated lymphocyte proliferation via a mechanism independent of IL-2 production, and caused a decrease in the expression and distribution of IL-2 receptors on the surface of responder cells.     

immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Primary structure of human alpha 2-macroglobulin. I. Isolation of the 26 CNBr fragments, amino acid sequence of 13 small CNBr fragments, amino acid sequence of methionine-containing peptides, and alignment of all CNBr fragments

The isolation of the 26 CNBr fragments from the identical Mr = 180,000 subunits of human alpha 2-macroglobulin is described. The fragments have been purified by combinations of gel chromatography, ion-exchange chromatography, high voltage paper electrophoresis, paper chromatography, and high performance liquid chromatography. The complete amino acid sequences of 13 small CNBr fragments have been determined. These fragments include CB1 (residues 1-9), CB3 (residues 79-98), CB4 (residues 99-128), CB9 (residues 442-477), CB10 (residues 478-497), CB13 (residues 644-650), CB14 (residues 651-665), CB15 (residues 666-674), CB16 (residues 675-690), CB19 (residues 937-945), CB20 (residues 946-954), CB24 (residues 1356-1362), and CB25 (residues 1363-1375). The fragments determined account for 200 of the 1451 residues of the subunits of alpha 2-macroglobulin. Most likely, Cys-6 of CB9 is bound to the corresponding residue in CB9 from another subunit, thus forming an interchain disulfide bridge in alpha 2-macroglobulin. Cys-1 of CB15 is bound to Cys-35 of CB12. CB15 contains a pair of Gln residues that can react covalently with amines in a factor XIIIa-catalyzed process (Gln-5 and Gln-6). CB16 contains the primary cleavage sites for proteinases in the bait region of alpha 2-macroglobulin (-Arg7-Val-Gly-Phe-Tyr-Glu-). CB20 contains the residues which in native alpha 2-macroglobulin presumably form an internal reactive beta-cysteinyl-gamma-glutamyl thiol ester (Cys-4 and Glx-7). Partial NH2- and COOH-terminal sequence data are given for the 13 large CNBr fragments. Complete or partial sequence determination of 19 methionine-containing peptides or variants thereof allow the alignment of all the CNBr fragments.     

Two proteolytic degradation products of calf-thymus poly(ADP-ribose) polymerase are efficient ADP-ribose acceptors. Implications for polymerase architecture and the automodification of the polymerase

Two polypeptides with molecular masses of 76 and 59 kDa were found to copurify with poly(ADP-ribose) polymerase from calf thymus, and to be as efficient acceptors of ADP-ribose as the polymerase itself. Analysis of their CNBr fragments by sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed that the polypeptides were derived from the 112-kDa polymerase. Isolation of poly(ADP-ribose) polymerase in the absence of protease inhibitors resulted in a loss of more than 90% of the polymerase activity and an increased proportion of the 76-kDa and 59-kDa polypeptides in the final polymerase preparation. When the polymerase and the two polypeptides were separated by gel filtration or polyacrylamide gel electrophoresis in 5% acetic acid, no polymerase activity was found associated with the two fragments. Analysis of the CNBr fragments of the three polypeptides after incubation of the enzyme preparation with [32P]NAD showed that most of the fragments were radioactive, indicating multiple ADP-ribosylation sites. Several ADP-ribosylated fragments were found to be common to all three polypeptides, or to two of them.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Summary Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by doublestaining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with M _r46000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Meiotic and morphological analysis in Hordeum pubiflorum (sect. Critesion, Triticeae)

Morphological and hybridization studies were carried out in H. pubiflorum s. 1. (2n= 14). Chromosome pairing observed at MI in the hybrids was high, but indications of weak sterility barriers were observed. It is concluded that (i) hybridization is fairly easy to perform, and the populations studied belong to the same species, (ii) no divergence in the ssp. halophilum genome was observed, except in (iii) a population with at least one reciprocal translocation, (iv) the halophilum × breviaristatum hybrids had lowered pairing with an increased frequency of univalents, (v) the pairing combined with morphology suggest recognition of H. pubiflorum ssp. breviaristatum (Parodi & Nicora) C. Baden (comb. nov.).     

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

PocketOptimizer 2.0: A modular framework for computer-aided ligand-binding design

The ability to design customized proteins to perform specific tasks is of great interest. We are particularly interested in the design of sensitive and specific small molecule ligand-binding proteins for biotechnological or biomedical applications. Computational methods can narrow down the immense combinatorial space to find the best solution and thus provide starting points for experimental procedures. However, success rates strongly depend on accurate modeling and energetic evaluation. Not only intra- but also intermolecular interactions have to be considered. To address this problem, we developed PocketOptimizer, a modular computational protein design pipeline, that predicts mutations in the binding pockets of proteins to increase affinity for a specific ligand. Its modularity enables users to compare different combinations of force fields, rotamer libraries, and scoring functions. Here, we present a much-improved version––PocketOptimizer 2.0. We implemented a cleaner user interface, an extended architecture with more supported tools, such as force fields and scoring functions, a backbone-dependent rotamer library, as well as different improvements in the underlying algorithms. Version 2.0 was tested against a benchmark of design cases and assessed in comparison to the first version. Our results show how newly implemented features such as the new rotamer library can lead to improved prediction accuracy. Therefore, we believe that PocketOptimizer 2.0, with its many new and improved functionalities, provides a robust and versatile environment for the design of small molecule-binding pockets in proteins. It is widely applicable and extendible due to its modular framework. PocketOptimizer 2.0 can be downloaded at https://github.com/Hoecker-Lab/pocketoptimizer .     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997