Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity.

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.

The product of the mos proto-oncogene as a candidate "initiator" for oocyte maturation.

The endogenous c-mos product, pp39mos, is required for progesterone-induced meiotic maturation in Xenopus oocytes. Treatment of oocytes with progesterone induced a rapid increase in pp39mos that preceded both the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). Microinjection of synthetic mos RNA into oocytes activated MPF and induced GVBD in the absence of progesterone. Thus, the mos proto-oncogene product may qualify as a candidate "initiator" protein of MPF and is at least one of the "triggers" for G2 to M transition.

Protection of mice against fatal herpes simplex type 2 infection by liposomes containing muramyl tripeptide.

Intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide that activates macrophages to a cytolytic state in situ, significantly protected mice against lethal challenge with herpes simplex virus type 2. These findings suggest that the systemic activation of macrophages by liposomes containing an immunomodulator can lead to prophylaxis of severe infections caused by herpesviruses.

Human monocytes activated by immunomodulators in liposomes lyse herpesvirus-infected but not normal cells.

Highly purified peripheral blood monocytes from normal human donors were activated in vitro by incubation with liposomes containing immunomodulators such as recombinant human gamma interferon, human lymphokines, or muramyl dipeptide. The ability of liposomes containing immunomodulators to activate monocytes to a cytotoxic state capable of discriminating between virus-infected and uninfected cells was shown by activated monocytes recognizing and destroying herpes simplex virus type 2-infected cells while leaving uninfected cells unharmed .

Characterization of chicken c-ski oncogene products expressed by retrovirus vectors.

We constructed replication-competent avian retrovirus vectors that contain two of the three known types of chicken c-ski cDNAs and a third vector that contains a truncated c-ski cDNA. We developed antisera that recognize the c-ski proteins made by the three transforming c-ski viruses. All three proteins (apparent molecular masses, 50, 60, and 90 kilodaltons) are localized primarily in the nucleus. The proteins are differentially phosphorylated; immunofluorescence also suggests that there are differences in subnuclear localization of the c-ski proteins and that c-ski protein is associated with condensed chromatin in dividing cells.

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

Herpes simplex virus-induced suppression of macrophage-mediated tumoricidal activity in murine macrophages.

Herpes simplex virus (HSV) infections can enhance the progression of neoplastic diseases. Since macrophages can be activated to become tumorilytic and may figure prominently in host defense against cancer, the ability of HSV to modify macrophage-mediated tumoricidal functions was evaluated. Murine peritoneal macrophages treated with HSV could not be activated to a tumoricidal state by mouse recombinant gamma-interferon (IFN-gamma). Addition of HSV 4 h after treatment with IFN-gamma, at a time when the macrophages are fully committed to developing the cytotoxic phenotype, blocked macrophage-mediated lysis of syngeneic melanoma target cells. This inhibition of activation and cytotoxicity was not due simply to uptake of virus particles, because treatment with heat-inactivated HSV at 4-h posttreatment with IFN-gamma had no effect. In addition, HSV did not undergo a productive infection within macrophages, suggesting that the observed inhibitory activity might be due to a virus-induced product. In this regard, treatment of macrophages with recombinant alpha-interferon suppressed the activation of these cells by IFN-gamma, suggesting that virus-induced alpha-interferon may be mediating all or part of the suppressive activity. These studies suggest that enhancement of tumor progression following HSV infection may be related to the virus-induced suppression of macrophage-mediated tumoricidal activity.

Characterization and localization of the products of the human homologs of the v-ets oncogene.

The avian erythroblastosis virus, E26, an acute leukemia virus, contains a transforming gene composed of two cellular components, v-myb and v-ets. The v-ets related sequences of man and other mammals consist of two transcriptionally active genes, ets-1 and ets-2, located on separate chromosomes. By contrast, both of these genes are contiguous in birds, are located on the same chromosome, and are coordinately transcribed. The human ets-1 and ets-2 gene products were identified by means of antibodies directed against the ets-1 and ets-2 encoded products. A 51 kD protein has been identified as the ets-1 gene product, and a 56 kD protein as the ets-2 gene product. Cellular fractionation studies indicated that the ets-1 protein is located in the cytoplasm and the ets-2 protein is nuclear. By comparison, the chicken ets protein, which contains both the ets-1 and ets-2 domains, distributes equally between the cytoplasm and nucleus. The differential compartmentalization of the ets gene products and their non-coordinate expression suggest that these proteins have different biological functions.

Probing structure and function of the raf protein kinase domain with monoclonal antibodies.

Five monoclonal antibodies were generated against the raf kinase domain. All antibodies react with different isozymes of the raf family, as well as Raf proteins from different species, albeit with differential affinities. Epitope mapping showed all five epitopes clustered in the vicinity of the conserved APE sequence. Although thought to be an essential part of the catalytic site, antibody binding to that domain does not affect kinase activity in vitro or the capability to specifically associate with other cellular proteins. Based on a detailed dissection of the epitopes, a comparative analysis of secondary structure predictions indicates a common structural motif in that region, which is highly conserved amongst protein kinases of the serine/threonine as well as the tyrosine class.

High expression of ets-1 gene in human thymocytes and immature T leukemic cells.

The cellular ets-1 gene homologous to the 5' region of the v-ets sequence of the E26 retrovirus codes for a 6.8-kb mRNA that is translated into a 51-kDa protein in human cells. A survey of mRNA from human tissues showed the thymus as the tissue with the highest level of ets-1 transcription, within other hematopoietic organs and tissues, including spleen, fetal liver, lymph nodes, bone marrow, and peripheral lymphocytes exhibiting low or undetectable levels of hybridization. A high level of ets-1 expression was found in murine thymocyte mRNA as well. Investigation of the ets-1 expression levels in human leukemic samples showed that primary malignant T cells (T-ALLs), corresponding to intrathymic stages of maturation, have a much higher level of ets-1 mRNA than malignant T lymphoid cells with a mature phenotype, such as adult T cell leukemias (ATLs). T-ALLs were also higher in ets-1 expression than the other lymphoid (pre-T-ALL, c-ALL, pre-B-ALL) malignant cells analyzed. Insignificant amounts of the specific ets-1 mRNA were detected in several acute myeloid leukemias representing various degrees of maturation. The elevated ets-1 mRNA in thymocytes suggests a biological role for the ets-1 product in these cells that could be explored to investigate ets-1 function. Finally, the exhibited expression of ets-1 in lymphoid cells and absence from malignant myeloid cells makes it a candidate marker for phenotyping human hematopoietic tumors.

Human monocyte-mediated cytotoxicity against herpes simplex virus-infected cells: activation of cytotoxic monocytes by free and liposome-encapsulated lymphokines.

Human peripheral blood monocytes were incubated with free or liposome-encapsulated human lymphokines containing macrophage-activating factor (MAF) and tested for their effect on herpes simplex virus (HSV)-infected target cells. Activated monocytes lysed allogeneic HSV type 2 (HSV-2)-infected whole human embryo cells and xenogeneic BALB/c mouse embryo cells (10E2) without any significant effect on uninfected cells, as measured by release of 51Cr from target cells after 18 h of cocultivation. Kinetic studies revealed that lysis of virus-infected cells occurred by 10 h following cocultivation with activated monocytes. The inability of free MAF or supernatants from MAF-activated monocytes to lyse HSV-2-infected cells suggested that direct monocyte-target cell contact is required for monocyte-mediated cytotoxicity of the virus-infected cells. Monocytes activated with MAF suppressed the production of HSV-2 and HSV-1 from virus-infected cells more than control monocytes did. In addition, monocytes treated with liposome-encapsulated MAF selectively destroyed HSV-2-infected cells but left uninfected cells unharmed. The capacity of liposome-encapsulated immunomodulators to activate human monocytes to selectively lyse HSV-2-infected cells has potential therapeutic benefit and should be evaluated in vivo.

Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence.

Human monocyte-derived neutrophil chemotactic factor (MDNCF) was purified from culture supernatant of lipopolysaccharide-stimulated human peripheral blood mononuclear leukocytes on a column of Sepharose-bound murine monoclonal anti-MDNCF. About 65% of the culture fluid chemotactic activity was bound to the column. The unbound 35% probably represents chemotactic activity of other cytokines in the culture fluid. More than 85% of the bound activity was eluted by pH 2.5 glycine buffer. When this material was applied to an HPLC-CM column, gradient elution produced four well-separated A280 peaks, each of which had chemotactic activity. N-terminal amino acid analysis of the four peaks revealed three different sequences. One (MDNCF-c) was identical to the sequence that we reported previously. The other two (MDNCF-a and -b) had seven and five additional amino acids, respectively, at the N-terminus. MDNCF-a, -b and -c accounted for 8, 47 and 45% of the total MDNCF peptide. Alignment with the MDNCF cDNA sequence shows that MDNCF-a results from cleavage of a 20 residue signal peptide. MDNCF-c results from culture fluid proteolytic cleavage of the N-terminal sequences of MDNCF-a and -b at an R-S bond. The three peptides occurred in the four HPLC-CM peaks in different ratios. The bulk of any one peptide was distributed in two adjacent HPLC-CM peaks. This suggests that each peptide exists in a minimum of two states. In contrast to our previous multi-step purification, the immunoaffinity and HPLC-CM column sequence resulted in complete purification of MDNCF in two steps and led to identification of two additional MDNCF peptides, one of which has not heretofore been detected.

Bacterial expression and characterization of nine polypeptides encoded by segments of the envelope gene of human immunodeficiency virus.

Nine envelope (Env) polypeptides, encoding different regions of HIV gp120 and gp41 Env proteins, and accounting for approx. 96% of the entire Env precursor glycoprotein complex (gp160) were expressed in Escherichia coli at levels ranging from approx. 2 to 20% of total cellular protein. The recombinant polypeptides were produced either as hybrid products fused to the cII gene fragment of the lambda vector or in an unfused form without interfering cII products. Partially purified protein fractions of each polypeptide were characterized serologically by Western-blot analysis against a panel of well characterized human immunodeficiency virus (HIV)-positive human reference sera. Most of the Env polypeptides were highly immunoreactive with anti-gp120/gp41 antibodies present in the sera of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related diseases, but the patterns of reactivity were different. These results demonstrate that some of the antigenic determinants residing on the viral gp160 complex are retained on the surfaces of the recombinant Env polypeptides, and suggest that these sites are differentially immunogenic. These results are therefore interpreted in the context of an ongoing process towards using bacterially expressed HIV Env polypeptides to help define biological and structural epitopes to aid in the development of more sensitive diagnostic and therapeutic reagents in the fight against AIDS.

Detection of heterologous fusion proteins in Escherichia coli with a monoclonal antibody.

Several laboratories have constructed expression vectors for the production of heterologous fusion proteins containing the N-terminal 13 amino acids of the bacteriophage lambda cII-coded protein in Escherichia coli. We have prepared a monoclonal antibody to a synthetic peptide having this CII amino acid sequence and have found that this antibody reacts with authentic CII protein in Western blot tests and with most CII peptide-containing fusion proteins in both radioimmunoprecipitation and Western blot assays. However, there are some CII-hybrid protein species with which the antibody does not react. Our findings indicate that this antibody is a valuable tool for detecting and purifying expressed proteins and in studying their structure and function.

Murine monoclonal antibodies which recognize active sites of Escherichia coli NusA protein and epitope mapping by gene fusion.

Seven hybridomas producing murine monoclonal antibodies reactive against NusA protein of Escherichia coli were prepared. Antigenic determinants of these monoclonal antibodies have been mapped by immunoblotting analyses using fusion proteins containing parts of NusA. The epitope of the N14 antibody maps in a hydrophobic amino acid (aa) cluster and consists of at least Ala-181 and Ser-183 residues. nusA1 and nusA11 mutations, which cause aa changes of these residues, abolish the antigenic reactivity to the N14 antibody. These antibodies react with intact NusA protein, indicating that the epitopes are exposed on the surface of NusA. Most of these epitopes cluster around the nusA1 and nusA11 mutation loci.

Inhibition of human immunodeficiency virus (HIV-1) replication in vitro by noncytotoxic doses of camptothecin, a topoisomerase I inhibitor.

We examined the effects of topoisomerase inhibitors on human immunodeficiency virus type 1 (HIV-1) infection of H9 cells in cell culture. Infection is blocked or substantially reduced by the topoisomerase I inhibitor camptothecin (CPT), but not by two topoisomerase II inhibitors. Significant reduction (greater than or equal to 90%) in the amount of virus released, as measured by reverse transcriptase, is obtained if the cells are treated for 1 h with 0.01-0.02 microM CPT at the time of virus infection, and expression of viral proteins is also blocked. CPT is also shown to reduce the level of infection when chronically infected cells are cocultivated with uninfected cells. These results with CPT suggest that this compound may represent a new class of drugs with antiretroviral potential.

Comparative analysis of gp41 antigens by enzyme-linked immunosorbent assays for detecting antibodies to human immunodeficiency virus type 1.

We have compared the antigenic qualities of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein with a synthetic oligopeptide (peptide R21S) and a bacterially synthesized protein (protein 566), which are homologous with the N-terminal region of gp41, in enzyme-linked immunosorbent assays (ELISA) for detecting antibodies to HIV-1 in sera of patients with the acquired immunodeficiency syndrome (AIDS) or the aids-related complex (ARC). Although the use of all three types of antigens readily allowed the detection of antibodies in human sera, ELISA employing purified gp41 glycoprotein and the protein 566 were more specific and sensitive than the peptide R21S ELISA.

Partial purification of native HIV transmembrane protein gp41: generation of polyclonal and monoclonal antibodies.

We partially purified the human immunodeficiency virus (HIV) glycoprotein gp41 from infected H9 cells by immunoaffinity chromatography using a column containing the M25 monoclonal antibody (diMarzo-Veronese et al., 1985). A pH 11.5 buffer worked best for eluting the glycoprotein from this column. The eluted gp41 was used in a sensitive slot blot immunoassay to detect antibodies to HIV in human sera and to prepare rabbit polyclonal antibodies and the 41-1S mouse monoclonal antibody. These antibodies reacted with gp41 in immunoprecipitation and in Western blot assays, but did not neutralize HIV in a syncytium-forming microassay. A pH 2.5 buffer was found to be the most effective solution for eluting gp41 from a 41-1S monoclonal antibody column.

Expression and purification of protein segments encoded by the envelope and 3'-orf genes of human immunodeficiency virus type 1.

The pJL6 expression vector and its derivatives, pJLA16 and pANH-1, have been used for the synthesis and high-level expression in Escherichia coli of restriction enzyme fragments derived from the envelope and 3'-orf genes of the BH10 and BH8 clones, respectively, of the human immunodeficiency virus (HIV-1). These bacterially expressed proteins have been purified to apparent homogeneity by sequential detergent extraction, gel filtration, and reverse-phase high-performance liquid chromatography. The recombinant proteins have been used for the production of polyclonal and monoclonal antibodies, and the fusion proteins from the envelope gene are currently being evaluated for use as immunodiagnostic assay reagants.

Purification of an Escherichia coli-expressed Nef protein from the human immunodeficiency virus-type 2.

The entire nef gene sequence of HIV-2, NIH-Z strain, has been cloned into the pJL6 expression vector and used for the synthesis of a 23-kDa protein in E. coli. The expressed protein is a fusion between the N-terminal 13 amino acids of the cII gene, 8 amino acids resulting from the ligation procedure, and the 180 amino acids that comprise the HIV-2 Nef sequence from the NIH-Z strain. The bacterially expressed Nef protein has been purified to apparent homogeneity on analytical scale (10-20 micrograms) by a combination of sequential detergent extraction, gel filtration, and reversed-phase high-performance chromatography. The expressed Nef protein is highly susceptible to proteolysis (chymotryptic-like activity) and this property accounts for the low yield obtained by gel filtration and RP-HPLC. Larger amounts (> 100 micrograms) of the purified Nef protein have been produced by a purification procedure that employs sequential detergent extraction, chromatography on Q-Sepharose in the presence of 7 M urea, and chromatography on hydroxylapatite, also in 7 M urea. The purified HIV-2 Nef protein has been used for the production of polyclonal and monoclonal antibodies. The milder method of purification should facilitate structure-function studies of the Nef protein and its role in the life cycle of HIV.