Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

Characterization of oligosaccharide chains on mouse mammary tumor virus envelope proteins and the implications for the mechanism of their glucocorticoid regulated processing

Glucocorticoid hormone is required for complete posttranslational processing of the glycosylated mouse mammary tumor virus envelope precursor, Pr74env in the murine T-lymphosarcoma cell line, W7MG1. Metabolic labeling studies with [35S]methionine, [3H]galactose, and [3H]mannose, combined with enzymatic digestion analyses with a variety of endoglycosidases, demonstrated that both proteolytic processing and N-linked oligosaccharide maturation depended, either directly or indirectly, on glucocorticoid action. Pr74 is found in both control and hormone-treated cells. In both cases Pr74 molecules carry high mannose and/or hybrid, but not complex, oligosaccharide chains with very little or no sialic acid. When cells are grown with glucocorticoid, Pr74 is converted to gp52 and gp33 with greatly increased efficiency, and these mature glycoproteins carry complex oligosaccharides containing sialic acid. No O-linked carbohydrate was detected on any of these species. According to this evidence, the glucocorticoid-regulated step in this pathway must occur at or before the final mannose trimming step in the Golgi that is required for formation of complex carbohydrate chains.     

Potent enzyme-activated inhibition of aromatase by a 7 alpha-substituted C19 steroid

Enzyme-activated inhibitors of aromatase would result in effective medicinal agents for modulating estrogen-dependent processes and thus may be useful in controlling reproductive processes and in treating estrogen-dependent diseases such as breast and endometrial cancer. A potential enzyme-activated inhibitor of aromatase, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD), was synthesized and examined in vitro with placental aromatase. Under initial velocity conditions, 7 alpha-APTADD exhibited high affinity for the enzyme and is a potent inhibitor of aromatase with an apparent Ki of 9.9 +/- 1.0 nM and with a Km for androstenedione of 52.5 +/- 5.9 nM. This inhibitor produced a rapid time-dependent, first-order inactivation of aromatase in the presence of NADPH, while no inactivation of aromatase activity was observed in the absence of NADPH. Protection of aromatase from inactivation was observed when the substrate, androstenedione, was included in the incubation mixture containing enzyme, inhibitor, and NADPH. On the other hand, nucleophilic trapping agents such as cysteine did not protect the enzyme from inactivation by 7 alpha-APTADD. Additionally, second enzyme pulse experiments demonstrated identical rates of inactivation, suggesting that the enzyme-activated inhibitor was not being released from the active site of the enzyme. The apparent Kinact for 7 alpha-APTADD is 159 +/- 21 nM and represents the inhibitor concentration required to produce a half-maximal rate of inactivation. The half-time of inactivation at infinite inhibitor concentration was 1.38 +/- 0.92 min and is the most rapid enzyme-activated aromatase inhibitor reported to date. Thus, 7 alpha-APTADD is a potent enzyme-activated inhibitor of aromatase, exhibiting high affinity and rapid inactivation. This inhibitor will be useful in probing the biochemistry of aromatase and should also serve as an effective medicinal agent for the treatment of estrogen-dependent cancers.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.     

Lysis of mouse macrophages, fibroblasts, and epidermal cells by epidermal alloantigen-specific cytotoxic T lymphocytes: effect of culture and inflammatory agents on Epa-1 expression

The expression of Epa-1, a tissue-restricted non-major histocompatibility complex (MHC) alloantigen, on CBA epidermal cells (EC), fibroblasts (FB), and macrophages (M phi) was investigated using bulk-cultured and clonally-derived anti-Epa-1 cytotoxic T lymphocytes (CTL). Epa-1 was readily detected on freshly trypsinized and 24-hr-cultured EC, and on skin FB cultured for 1-3 weeks. In contrast, fresh peritoneal (PE) M phi were specifically resistant to Epa-1 CTL but became susceptible after 12-24 hr in culture. Epa-1 expression by PE M phi also could be induced in vivo by M phi-activating agents such as concanavalin A or Bacillus Calmette-Guérin (BCG), but not by the sterile inflammatory agents peptone broth or thioglycolate, suggesting a correlation between Epa-1 phenotype and M phi activation. From this and from parallel studies of spleen cell M phi it is concluded that Epa-1 may be a strain-specific marker for activated M phi in the mouse, as well as an inducible histocompatibility antigen in vivo.     

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficiency of antigen presentation after antigen targeting to surface IgD, IgM, MHC, Fc gamma RII, and B220 molecules on murine splenic B cells

Bispecific heteroconjugate antibodies can bind soluble protein Ag to APC and thereby enhance Ag presentation. We used such antibodies to bind hen egg lysozyme (HEL) to various structures on the surface of normal splenic B cells to determine which structures would provide the best targets for enhanced presentation. We found that HEL was presented efficiently to hybridoma T cells if bound to sIgD, sIgM, or class I or II MHC molecules, but not at all if bound to Fc gamma RII, or B220 molecules on B cells. The efficiency of presentation of HEL was measured as a function of the amount of 125I-HEL bound per cell. HEL was presented with 5 to 10 times greater efficiency when bound to sIg, than when bound to MHC molecules. When compared on the basis of the amount of HEL bound, sIgD and sIgM functioned equally as target structures, as did class I and class II MHC molecules. Large amounts of HEL bound to B220, but no presentation resulted, indicating that focusing HEL to the APC surface was not sufficient for presentation to occur. HEL was internalized rapidly and in large amounts when bound to sIgD or sIgM, but slowly and in small amounts, when bound to class I or class II MHC molecules. Thus, a rapid rate of internalization may in part explain the high efficiency of Ag presentation after binding to sIg. However, the small amount of HEL internalized via MHC molecules was utilized efficiently for presentation. These results indicate that sIgM and sIgD serve equally on normal B cells to focus and internalize Ag and enhance Ag presentation, but that class I or class II MHC molecules can also be used to internalize Ag and enhance Ag presentation, perhaps by a separate intracellular processing pathway.     

From the Ground Up: Global Nitrous Oxide Sources are Constrained by Stable Isotope Values

Rising concentrations of nitrous oxide (N₂O) in the atmosphere are causing widespread concern because this trace gas plays a key role in the destruction of stratospheric ozone and it is a strong greenhouse gas. The successful mitigation of N₂O emissions requires a solid understanding of the relative importance of all N₂O sources and sinks. Stable isotope ratio measurements (δ¹⁵N-N₂O and δ¹⁸O-N₂O), including the intramolecular distribution of ¹⁵N (site preference), are one way to track different sources if they are isotopically distinct. ‘Top-down’ isotope mass-balance studies have had limited success balancing the global N₂O budget thus far because the isotopic signatures of soil, freshwater, and marine sources are poorly constrained and a comprehensive analysis of global N₂O stable isotope measurements has not been done. Here we used a robust analysis of all available in situ measurements to define key global N₂O sources. We showed that the marine source is isotopically distinct from soil and freshwater N₂O (the continental source). Further, the global average source (sum of all natural and anthropogenic sources) is largely controlled by soils and freshwaters. These findings substantiate past modelling studies that relied on several assumptions about the global N₂O cycle. Finally, a two-box-model and a Bayesian isotope mixing model revealed marine and continental N₂O sources have relative contributions of 24–26% and 74–76% to the total, respectively. Further, the Bayesian modeling exercise indicated the N₂O flux from freshwaters may be much larger than currently thought.     

Lysis and fractionation of Mycobacterium paratuberculosis and Escherichia coli by matrix solid-phase dispersion.

A novel method for the lysis and subsequent fractionation of bacterial constituents from Mycobacterium paratuberculosis strain 19698 (M. paratuberculosis) and Escherichia coli strain DH5 alpha utilizing the technique of matrix solid-phase dispersion (MSPD) is described. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain cellular lysis. The blended material was used to prepare a column which was sequentially eluted with solvents of increasing polarity. Fractionation of cellular components was confirmed by analysis of the solvent extracts. The possible applicability of the MSPD technique as a general method for the lysis and fractionation of bacterial components is proposed.