Direct and indirect evidence for polypeptide absorption by the teleost gastrointestinal tract

The ability of the gastrointestinal tract of chinook salmon, Oncorhynchus tshawytscha , to absorb polypeptides in vivo was investigated by reference to the appearance of orally administered adrenocorticotropic hormone (ACTH) within the blood of fish previously treated with dexamethasone (3μg g^−l body weight) in order to suppress endogenous ACTH secretion. Further, since cortisol presence within plasma is dependent upon the availability of ACTH, dexamethasone blockade of endogenous ACTH secretion, in conjunction with subsequent measurements of plasma cortisol levels, provides a means by which biological patency of absorbed exogenous ACTH may be demonstrated. Levels of ACTH and cortisol in plasma of dexamethasone-treated salmon were therefore measured for a period of 360 min immediately following oral intubation of ACTH (15μg g⁻¹ body weight). Peak plasma presence of ACTH-like immunoreactivity (676.6 ± 33.6 pg ml⁻¹ plasma) and cortisol (227.1 ± 29.0 ng ml⁻¹ plasma) were recorded 120 min after ACTH administration. Results from the experimental groups were compared to those of 15 control treatments. Since administration of ACTH to chinook salmon elicited a consistent and significant elevation in not only plasma ACTH but also cortisol presence, it is contended that the salmonid gut expresses an ability to absorb polypeptides of dietary origin. The significance of these findings with respect to the oral administration of biologically active peptides and proteins to fish of importance to aquaculture is discussed.     

The Fc-receptor III of cultured human monocytes. Structural similarity with FcRIII of natural killer cells and role in the extracellular lysis of sensitized erythrocytes

FcRIII is not present on peripheral blood monocytes, but becomes expressed upon culturing and can be demonstrated on tissue macrophages. We studied the expression of FcRIII of cultured monocytes in detail and compared its structure with FcRIII of neutrophils and NK cells. The cell density of FcRIII reached a plateau within 3 days of culturing. During that time, the expression of FcRI and FcRIIa, also present on monocytes, did not change significantly. FcRIII on cultured monocytes lacked, as did NK cell FcRIII, the NA1-allotypic variant of the NA system present on the neutrophil FcRIII. Studies with glycosyl-phosphatidyl-inositol-specific phospholipase C and analysis of cells of patients with paroxysmal nocturnal hemoglobinuria revealed that FcRIII on cultured monocytes is not anchored by phosphatidyl-inositol-glycan in the cell membrane. Similarly, FcRIII on NK cells was resistant to glycosyl-phosphatidyl-inositol-specific phospholipase C treatment, suggesting that NK cell FcRIII is also not anchored by a phosphatidyl-inositol-glycan moiety, in contrast to neutrophil FcRIII. Analysis by SDS-PAGE showed that the FcRIII of cultured monocytes had a similar mobility as the FcRIII on NK cells, but was clearly distinct from neutrophil FcRIII. Treatment with N-glycanase showed that the protein backbone of deglycosylated FcRIII of cultured monocytes was similar to that of FcRIII of NK cells, but deglycosylated neutrophil FcRIII was different. Specific blocking of FcRIII of cultured monocytes with an anti-FcRIII mAb did not reduced the lytic action of the cultured monocytes towards sensitized erythrocytes. However, FcRIII was modulated from the cell surface by incubation with sensitized E, whereas non-FcR Ag were not. These findings indicate that FcRIII is involved in binding of immune complexes, but does not act as a trigger molecule for extracellular lysis of sensitized E.     

Genetic identification of non-specific esterases of the mouse cauda epididymidis and description of esterase-28, a new carboxylesterase isoenzyme (EC 3.1.1.1)

Twenty-five (25) electrophoretic bands with esterase activity were distinguished in supernatants of cauda epididymidis of DBA/2J mice. Twenty (20) of these were assigned to 10 genetically defined esterases (ES-1, ES-2, ES-3, ES-6, ES-7, ES-11, ES-14, ES-17, ES-19, ES-22) which were already known from investigations of other mouse tissues. Furthermore, ES-10 was identified in cauda supernatants after isoelectric focussing. A hitherto genetically undefined esterase was assigned to locus Es-28 which was expressed solely in the epididymis. Three phenotypes were distinguished: ES-28A was present in the majority of the inbred strains examined. ES-28B was observed in AKR/Han mice and ES-28C was found in SEG/1 mice.     

Crystals of wild-type, mutated, derivatized and complexed 50 S ribosomal subunits from Bacillus stearothermophilus suitable for X-ray analysis

Three-dimensional single crystals of wild-type and mutated 50 S ribosomal subunits from Bacillus stearothermophilus, as well as crystals of reconstituted subunits containing heavy-atom clusters and complexes of these subunits with tRNA and a short nascent polypeptide chain, were grown from polyethylene glycol in the presence of salts at low concentrations. Within experimental error, all these crystals are isomorphous, packed with monoclinic symmetry (C2) in unit cells of a = 300 A, b = 546 A, c = 377 (+/- 1%) A and beta = 112 degrees. Using synchrotron radiation at 85 to 100 K they diffract to 11 A resolution and can be irradiated for hours without disintegrating, so that a complete data set could be collected from a single crystal.     

Modification of tumor cells by a low dose of Newcastle disease virus

Summary Augmented tumor-specific T cell responses were observed against the high metastatic murine lymphoma variant ESb when using as immunogen ESb tumor cells that had been modified by infection with a low dose of Newcastle disease virus (NDV). Such virus-modified inactivated tumor cells (ESb-NDV) were potent tumor vaccines when applied postoperatively for active specific immunotherapy of ESb metastases. We demonstrate here that immune spleen cells from mice immunized with ESb-NDV contain enhanced immune capacity in both the CD4⁺, CD8^– and the CD4^–, CD8⁺ T cell compartments to mount a secondary-tumor-specific cytotoxic T cell response in comparison with immune cells from mice immunized with ESb. ESb-NDV immune CD4⁺, CD8^– helper T cells also produced more interleukin 2 after antigen stimulation than the corresponding ESb immune cells. There was no participation of either CD4⁺ or CD8⁺ virus-specific cells in the augmented response. The specificity of the T cells for the tumor-associated antigen remaind unchanged. Thus, there is the paradox that the virus-mediated augmentation of the tumor-specific T cell response in this system involves increased T helper activity but does not involve the recognition of viral epitopes as potential new helper determinants.Abbreviations CTL cytolytic T lymphocytes - IL-2 interleukin 2 - rIL-2 recombinant IL-2 - mAb monoclonal antibody - NDV Newcastle disease virus - SSC syngeneic spleen cell     

Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus

The satellite RNA of tobacco ringspot virus (sTobRV) undergoes self-catalysed cleavage during replication. A plasmid for in vitro expression of sTobRV has been constructed and used to obtain a library of mutagenized sTobRV sequences. Screening of these mutants has allowed precise definition of the sequences required for (+) and (-) strand cleavage. The sequences and RNA structures associated with cleavage of each strand differ markedly. Cleavage of the (+) strand requires those sequences flanking the site for cleavage to form a 'hammerhead' domain, similar to those found in other satellite and viroid RNA. In contrast, cleavage of the (-) strand requires only a small region of 12 nucleotides (nt) at the site of cleavage, and a sequence of 55 nt positioned elsewhere in the molecule. Comparison with a closely related satellite suggests that a novel RNA structure may be involved in (-) strand cleavage.     

delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase

A multienzyme catalyzing the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the first free intermediate in penicillin biosynthesis, was detected in an assay measuring the formation of tripeptide from L-[U-14C]valine in the presence of L-alpha-aminoadipic acid, L-cysteine, ATP, Mg2+ ions, and dithioerythritol. Enzyme was extracted from dry mycelium using a buffer with a high glycerol concentration and thiol protective agent to stabilize enzyme activity. In five steps the enzyme was purified 118-fold. It catalyzed ATP-pyrophosphate exchange in dependence of all three constituent amino acids, and the enzyme could be amino-acylated with L-[14C]valine. The molecular weight of the protein both native (in gel filtration chromatography) and denatured (polyacrylamide gel electrophoresis) was about 220 kDa. These data suggest that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase consists of a single polypeptide chain and a multienzyme thiotemplate mechanism for the reaction sequence is postulated.     

Structural and enzymatic studies of the T4 DNA replication system. I. Physical characterization of the polymerase accessory protein complex

In this study, we have investigated the structural and physical properties of the bacteriophage T4 DNA polymerase accessory proteins. We find that T4 gene 44 and 62 proteins associate to form a tight, highly homogeneous complex, containing four gene 44 protein subunits and one gene 62 protein subunit. The molecular mass of the complex is 163,700 daltons. Sedimentation results suggest that the complex is quite asymmetric, with a prolate ellipsoid axial ratio of about 5:1. This protein complex is known to carry a DNA-dependent ATPase activity; we show by photoaffinity labeling that the ATP-binding sites reside in the gene 44 protein subunits of the complex. Equilibrium sedimentation and chemical cross-linking studies indicate that the T4 gene 45 protein self-associates to form a trimer in solution. This trimer species also appears to be quite asymmetric, showing an axial ratio for a prolate ellipsoid of about 6:1, assuming normal hydration.     

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.