Conformation of the transmembrane domain of the c-erbB-2 oncogene-encoded protein in its monomeric and dimeric states

The human c-erbB-2 oncogene is homologous to the ratneu oncogene, both encoding transmembrane growth factor receptors. Overexpression and point mutations in the transmembrane domain of the encoded proteins in both cases have been implicated in cell transformation and carcinogenesis. In the case of theneu protein, it has been proposed that these effects are mediated by conformational preferences for anα-helix in the transmembrane domain, which facilitates receptor dimerization, an important step in the signal transduction process. To examine whether this is the case for c-erbB-2 as well, we have used conformational energy analysis to determine the preferred three-dimensional structures for the transmembrane domain of the c-erbB-2 protein from residues 650 to 668 with Val (nontransforming) and Glu (transforming) at position 659. The global minimum energy conformation for the Val-659 peptide from the normal, nontransforming protein was found to contain several bends, whereas the global minimum energy conformation for Glu-659 peptide from the mutant, transforming protein was found to beα-helical. Thus, the difference in conformational preferences for these transmembrane domains may explain the difference in transforming ability of these proteins. The presence of higher-energyα-helical conformations for the transmembrane domain from the normal Val-659 protein may provide an explanation for the presence of a transforming effect from overexpression of c-erbB-2. In addition, docking of the oncogenic sequences in theirα-helical and bend conformations shows that the all-α-helical dimer is clearly favored energetically over the bend dimer.     

Effect of tumor necrosis factor-α and interferon-γ on the growth of a human salivary gland cell line

Interferon-γ (IFN-γ) is a product of activated T-lymphocytes, and tumor necrosis factor-α (TNF-α) is a product of both lymphocytes and macrophages. These cell types are often present at sites of tissue damage secondary to chronic infection or autoimmune disease. The purpose of this study was to characterize the effects of TNF-α and IFN-γ on a human submandibular gland epithelial cell line (HSG). IFN-γ caused a concentration-dependent decrease in HSG cell growth (～70% in 6 days). Conversely, TNF-α alone had little effect on the growth of these cells. When these cytokines were added in combination (20 units/ml TNF-α and 1,000 units/ml of IFN-γ), there was a synergistic antiproliferative effect; no apparent cell growth was observed. The cytokine-induced antiproliferative effect was reversible. After the apparent cessation of cell growth for 3–6 days, removal of the cytokines permitted complete growth recovery. Further, cells that recovered and exhibited growth patterns that were similar to control cells remained susceptible to the antiproliferative effects of the cytokines. Flow cytometry revealed that the percentage of cells in G0/G1 with the combination of cytokines was significantly increased by 24 h. The antiproliferative effect of IFN-γ alone and that of IFN-γ and TNF-α in combination were blocked completely using an antibody to the IFN-γ receptor. A hypothesized mechanism of tissue damage in autoimmune inflammatory disorders is via up-regulation of cell surface markers such as intercellular adhesion molecule type I (ICAM-1) and histocompatibility antigen HLA-DR which can exacerbate the inflammatory process. Treatment of HSG cells with IFN-γ, with or without TNF-α, resulted in increased levels of ICAM-1 and the acquisition of HLA-DR expression. These aggregate data suggest that IFN-γ alone can regulate the expression of cell surface markers involved in the inflammatory process as well as cause a potent yet reversible inhibition of HSG cell growth that is modulated by the presence of TNF-α. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.     

Purification,and characterization of a β-mannanase of Trichoderma reesei C-30

Trichoderma reesei was studied for its ability to produce -mannanase activity on a variety of carbon sources. The highest -mannanase activity was produced on cellulose, whereas -mannan-containing carbon sources (such as kojac powder or locust bean gum) gave lower enzyme titres. The enzyme responsible for the major -mannanolytic activity from T. reesei was purified to physical homogeneity by preparative chromatofocusing and anion exchange fast protein liquid chromatography. This -mannanase is a glycoprotein, with a molecular mass of 46 (±2) kDa and an isoelectric point of 5.2. It has an optimal pH at 5.0 and broad pH stability (2.5–7.0). It is stable for 60 min at 55° C, and has an optimal temperature for activity at 75° C. During incubation with locust bean gum, the enzyme releases mainly tri- and disaccharides. Correspondence to: C. P. Kubicek     

Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.

The paraoxonase/arylesterase gene is located close to the cystic fibrosis gene on chromosome 7. Human serum contains two paraoxonase/arylesterase allozymes, A and B, which differ in their substrate specificities and kinetic properties. Purified A, AB, and B esterases were digested with trypsin, and the resultant peptides were compared by high-performance liquid chromatography. The elution profiles were very similar for all three samples, except for (1) one peptide (i.e., peptide A) seen only in the A and AB profiles and (2) another peptide (i.e., peptide B) seen only in the B and AB profiles. Sequencing revealed that peptide A had glutamine at amino acid position 191, whereas peptide B was generated by cleavage on the carboxy side of position 191, presumably because there was a basic (trypsin-specific) amino acid at that position. Working independently, our laboratory and one other laboratory have sequenced the coding region for paraoxonase from human liver cDNA libraries and have identified two polymorphic sites: Arg/Gln at position 191 and Leu/Met at position 54. Using PCR amplification and direct sequencing of nucleotides in both polymorphic regions with genomic DNA, we have estimated the allelic frequencies and have determined their concordance with the serum paraoxonase allozyme phenotypes in 27 unrelated adults and in 16 members of a three-generation pedigree. Among unrelated individuals, the Met/Leu polymorphism at position 54 did not correlate with the serum esterase phenotype. In contrast, the particular amino acid at position 191 correlated perfectly with serum phenotypes: A-type individuals had Gln at position 191, and B-type individuals had Arg at position 191; AB-type serum was found only with the heterozygous (Arg/Gln) combination. Pedigree analysis showed both polymorphisms to be inherited in the expected Mendelian manner and confirmed that only the 191 polymorphism showed concordance with the serum paraoxonase/arylesterase phenotypes.     

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.