Impaired local natural killer cell activity in human colorectal carcinomas

Summary The present study was undertaken to study natural killer (NK) cell activity in patients with colorectal cancer at peripheral and local levels. Mononuclear cells were isolated from uninvolved colorectal mucosa, tumor tissue and peripheral blood, and tested against the colon carcinoma cell line CaCo-2 and the erythroleukemia cell line K-562. Peripheral blood NK cell activity from the patients showed similar levels compared with healthy controls, whereas, mononuclear cells of tumor tissue were found to have a significantly decreased NK cell activity compared to the normal intestinal mucosa (P<0.01). No relation was found between the NK cell activity and the advancement of the disease according to the Duke's stage. Interferon- (IFN-) stimulated the NK cell activity of the mononuclear cells from blood, mucosa and tumor. However, the increase of NK cell activity after IFN- stimulation was lower in the tumor compared to the mucosa (P<0.02). The lectin, phytohaemagglutinin, increased the cytotoxicity of mononuclear cells from blood, mucosa and tumor to a similar level. These results suggest that patients with colorectal tumors exhibit a normal NK cell activity in peripheral blood and intestinal mucosa; however, a diminished NK cell activity exists at the tumor level. Although mononuclear cells isolated from the tumor have a normal response to lectin stimulation they show hyporesponsiveness to IFN- stimulation with regard to their NK cell activity.     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Denitrification and oxygen respiration in biofilms studied with a microsensor for nitrous oxide and oxygen

Depth distributions of O₂ respiration and denitrification activity were studied in 1- to 2-mm thick biofilms from nutrient-rich Danish streams. Acetylene was added to block the reduction of N₂O, and micro-profiles of O₂ and N₂O in the biofilm were measured simultaneously with a polarographic microsensor. The specific activities of the two respiratory processes were calculated from the microprofiles using a one-dimensional diffusion-reaction model. Denitrification only occurred in layers where O₂ was absent or present at low concentrations (of a fewM). Introduction of O₂ into deeper layers inhibited denitrification, but the process started immediately after anoxic conditions were reestablished. Denitrification activity was present at greater depth in the biofilm when the NO₃ ^– concentration in the overlying water was elevated, and the deepest occurrence of denitrification was apparently determined by the depth penetration of NO₃ ^–. The denitrification rate within each specific layer was not affected by an increase in NO₃ ^– concentration, and the half-saturation concentration (K_m) for NO₃ ^– therefore considered to be low (<25M). Addition of 0.2% yeast extract stimulated denitrification only in the uppermost 0.2 mm of the denitrification zone indicating a very efficient utilization of the dissolved organic matter within the upper layers of the biofilm.     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.     

Molecular cloning,nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

Summary The -cyclodextrin glycosyltransferase (-CGTase) gene was isolated from a -library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant -CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme. Offprint requests to: H. Bender     

Linker insertion analysis of the FimH adhesin of type 1 fimbriae in an Escherichia coli fimH-null background

Abstract The gene encoding the Escherichia coli FimH adhesin of type 1 fimbriae has been subjected to linker insertion mutagenesis. Amino acid changes were introduced at a number of positions spanning the entire sequence in order to probe the structure-function relationship of the FimH protein. The effect of these mutations on the ability of bacteria to express a D-mannose binding phenotype was assessed in a fimH null mutant (MS4) constructed by allelic exchange in the E. coli K-12 strain PC31. Mutations mapping at amino acid residues 36, 58 and 279 of the mature FimH protein were shown to completely abolish binding to D-mannose receptors. Differences in the level of fimbriation were also observed as a result of some of the mutations in the fimH gene. These mutants may prove useful in dissecting receptor-ligand interactions by defining regions of the FimH protein that are important in erythrocyte binding.     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Glutamine is not an essential amino acid for Sf-9 insect cells

Summary Spodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of -galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.