Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Molecular cloning of theEnvC gene ofEscherichia coli

DNA fragments complementing theenvC mutation could be isolated by cloning chromosomal DNA in the vector pUH84. When the frequencies of transformation and the frequencies of restoring theenvC ⁺ phenotype were compared, the high copy number hybrid plasmids complemented with a frequency of 10^–5. After subcloning theenvC-complementing DNA fragment into the low copy number plasmid pLG339, efficient complementation was achieved by spontaneous integration of the IS2 element ofEscherichia coli. By nucleotide sequence analysis, a potential promoter, a ribosome-binding site, and an unidentified reading frame were detected in the respective DNA fragment.     

Virus penetration of examination gloves

Examination gloves worn for protection from biohazards were sampled and evaluated for their ability to exclude virus particles. We found that thin gloves manufactured from polyethylene or polyvinyl chloride are ineffective barriers while gloves of thin latex are superior but not without failure. Polyethylene and polyvinyl chloride gloves had failure rates of 40% and 22%, respectively. Following exposure to the common disinfectant, 70% ethanol, these failure rates increased to 94% and 56% for polyethylene and polyvinyl chloride gloves, respectively. Latex, although permeable to ethanol, was penetrated by virus less than 1% of the time regardless of whether the latex had been pre-exposed to disinfectant or not. This study highlights the need for caution on the part of those who rely upon examination gloves for protection from infectious agents as well as the need for establishing more adequate standards and testing procedures for their manufacture.     

Structure and expression of the cAMP cell-surface receptor

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Rhizosphere microorganism effects on soluble amino acids,sugars and organic acids in the root zone ofAgropyron cristatum,A. smithii andBouteloua gracilis

Three axenic and rhizosphere microorganism-inoculated shortgrass steppe plant species were evaluated for possible differences in residual organic carbon and nitrogen present as sugars, organic acids and amino acids. IntroducedAgropyron cristatum was compared toA. smithii andBouteloua gracilis, which are dominant species in the native shortgrass steppe. These plants, grown for 90 days in root growth chambers, showed differences in residual organic carbon and nitrogen per gram of root, and rhizosphere microbe presence resulted in additional changes in these compounds. The root biomass ofB. gracilis was significantly increased with microbes present. TheAgropyron species had significantly lower amino acid levels with microbes present, while under the same conditions, theB. gracilis showed significant decreases in residual sugars. Based on the amino acids, sugars and organic acids, the C/N ratio of the sterileA. cristatum was higher than forB. gracilis. Rhizosphere microbe presence did not result in changes in these C/N ratios. These results suggest thatA. cristatum, with microbes present, will have lower levels of amino acids present, whileB. gracilis, with a lower C/N ratio, will have sugars used to a greater extent by the rhizosphere microbes. This resulted in the higher levels of residual soluble organic C and N in the rhizosphere ofB. gracilis, in comparison with the introducedA. cristatum. These differences may be critical in influencing the course of nutrient accumulation and plant competition in short-grass steppe communities, and in understanding basic aspects of plant-rhizosphere microorganism interactions.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

Molecular basis of growth cone adhesion: anchoring of adheron- containing filaments at adhesive loci

Adhesive contacts made by filopodia of neuronal growth cones are essential for proper neurite elongation and may have a role in the formation of synaptic junctions. Previously we described the appearance of filamentous materials extending from growth cone surfaces that seem to be associated with the strongly adhesive behavior of filopodia (Tsui, H.-C., K. L. Lankford, and W. L. Klein. 1985. Proc. Natl. Acad. Sci. USA. 82:8256-8260). Here, we have used immunogold labeling to determine whether known adhesive molecules might be localized at points of adhesion and possibly be constituents of the filamentous material. Antibodies to an adhesive molecule (neural cell adhesion molecule [N-CAM]) and to an adhesive macromolecular complex of proteins and proteoglycans (adheron) were localized at the EM level in whole mounts of cultured avian retina cells. Labeling of fixed cells showed that N-CAM and adheron molecules were both present on growth cones and on filopodia. However, filamentous materials extending from the cell surface were labeled with anti-adheron but not with anti-N-CAM. If cells were labeled before fixation, patches of anti-N-CAM labeling occurred in random areas over the growth cones, but adheron antibodies concentrated at points of apparent adhesion. Particularly dense clustering of anti-adheron occurred at individual filopodial tips and at points of contact between pairs of filopodia. The different patterns of labeling imply that N-CAMS do not associate with the main antigenic components of adheron on the membrane surface. Most importantly, the data indicate the N-CAMs were mobile in the membrane but that constituents of adherons were anchored at adhesive loci. An appealing hypothesis is that molecules found in adheron preparations have an important role in establishing the adhesive junctions formed by growth cone filopodia.     

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.