Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Effect of endotoxin on lipid order and motion in erythrocyte membranes

Electron paramagnetic resonance employing a lipid-specific spin label has been used to investigate the molecular effects of endotoxin on the physical state of bilayer lipids in rat erythrocyte membranes. When added at a concentration as low as 40 μg/ml to whole blood (plasma plus leukocytes present), decreased membrane lipid motion was found in subsequently washed and spin-labeled intact erythrocytes (P < 0.02). However, if endotoxin were added to washed, plasma plus leukocyte-free intact erythrocytes, no change in the motion of the spin label was found, suggesting that plasma-soluble substances and/or leukocytes are required to produce the change in the physical state of lipids. The decreased lipid motion found in these studies is discussed with reference to the known decreased deformability of endotoxin-treated red cells and to the pathogenesis of sepsis.     

Characterization of DNA helicase II from a uvrD252 mutant of Escherichia coli.

The loss of DNA helicase II (UvrD) in Escherichia coli results in sensitivity to UV light and increased levels of spontaneous mutagenesis. While the effects of various uvrD alleles have been analyzed in vivo, the proteins produced by these alleles have not been examined in any detail. We have cloned one of these alleles, uvrD252, and determined the site of the mutation conferring the phenotype. In addition, the protein it encodes has been purified to homogeneity and characterized in vitro. The mutation responsible for the phenotype was identified as a glycine-to-aspartic-acid change in the putative ATP-binding domain. In comparison to wild-type DNA helicase II, the UvrD252 enzyme exhibited reduced levels of ATPase activity and a large increase in the Km for ATP. The ability of UvrD252 to unwind DNA containing single-stranded regions, as well as DNA containing only nicks, was reduced in comparison to that of the wild-type enzyme. Possible interpretations of these results in relation to the phenotypes of the uvrD252 mutant are discussed. This represents the first detailed analysis of the biochemical properties of a mutant DNA helicase II protein.     

Reduction in animal abundance and oxygen availability during and after the end-Triassic mass extinction

The end-Triassic biodiversity crisis was one of the most severe mass extinctions in the history of animal life. However, the extent to which the loss of taxonomic diversity was coupled with a reduction in organismal abundance remains to be quantified. Further, the temporal relationship between organismal abundance and local marine redox conditions is lacking in carbonate sections. To address these questions, we measured skeletal grain abundance in shallow-marine limestones by point counting 293 thin sections from four stratigraphic sections across the Triassic/Jurassic boundary in the Lombardy Basin and Apennine Platform of western Tethys. Skeletal abundance decreased abruptly across the Triassic/Jurassic boundary in all stratigraphic sections. The abundance of skeletal organisms remained low throughout the lower-middle Hettangian strata and began to rebound during the late Hettangian and early Sinemurian. A two-way ANOVA indicates that sample age (p < .01, η² = 0.30) explains more of the variation in skeletal abundance than the depositional environment or paleobathymetry (p < .01, η² = 0.15). Measured I/Ca ratios, a proxy for local shallow-marine redox conditions, show this same pattern with the lowest I/Ca ratios occurring in the early Hettangian. The close correspondence between oceanic water column oxygen levels and skeletal abundance indicates a connection between redox conditions and benthic organismal abundance across the Triassic/Jurassic boundary. These findings indicate that the end-Triassic mass extinction reduced not only the biodiversity but also the carrying capacity for skeletal organisms in early Hettangian ecosystems, adding to evidence that mass extinction of species generally leads to mass rarity among survivors.     

Local helix content in an alanine-rich peptide as determined by the complete set of 3JHNα coupling constants

Summary Alanine-rich peptides serve as models for exploring the factors that control helix structure in peptides and proteins. Scalar CH-NH couplings (³J_HN) are an extremely useful measure of local helix content; however, the large alanine content in these peptides leads to significant signal overlap in the CH region of ¹H 2D NMR spectra. Quantitative determination of all possible ³J_HN values is, therefore, very challenging. Szyperski and co-workers [(1992) J. Magn. Reson., 99, 552–560] have recently developed a method for determining ³J_HN from NOESY spectra. Because ³J_HN may be determined from 2D peaks outside of the CH region, there is a much greater likelihood of identifying resolved resonances and measuring the associated coupling constants. It is demonstrated here that ³J_HN can be obtained for every residue in the helical peptide Ac-(AAAAK)₃A-NH₂. The resulting ³J_HN profile clearly identifies a helical structure in the middle of the peptide and further suggests that the respective helix termini unfold via distinct pathways.Abbreviations ³J_HN three-bond CH-NH scalar coupling constant - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser spectroscopy - COSY two-dimensional correlated spectroscopy - DQF-COSY two-dimensional double-quantum-filtered correlated spectroscopy - TOCSY two-dimensional total correlation spectroscopy To whom correspondence should be addressed.Deceased March 5, 1996.