Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Vaccines for parasitic diseases.

Significant effort and progress has occurred over the last several years in the development of vaccines against three main tropical parasitic diseases (malaria, leishmaniases and schistosomiasis). However, an effective vaccine is not yet available. The difficulties in developing a vaccine against parasitic disease are complicated not only by the necessity to identify (and produce) appropriate, protective antigens but also a lack of complete understanding of the types of immune responses needed for protection. Despite these hurdles, several candidate vaccines are under development for each disease; at least one promising vaccine candidate exists that is in late stage clinical testing.

The glucose effect in Bacillus subtilis.

An analysis of the glucose downshift mechanism in Bacillus subtilis has shown that the depression of catabolic enzymes characteristic of the 'glucose effect' includes isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. Additionally, phosphofructokinase undergoes what appears to be a reversible modification regulated by glucose transport.

A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.

Transcriptional inhibition and production of guanosine polyphosphates in Bacillus subtilis.

When exponentially growing cells of Bacillus subtilis were treated with rifampin or lipiarmycin, both inhibitors of the initiation of ribonucleic acid synthesis, large amounts of (p)ppGpp accumulated. This accumulation appears to be independent of the ribosome-dependent stringent factor reaction because both relA and relC mutants responded in a manner similar to that of the wild type. The possibility that ribonucleic acid polymerase is directly involved in (p)ppGpp metabolism is discussed.

Bacillus subtilis relG mutant: defect in glucose uptake.

A previously described Bacillus subtilis mutant which exhibits a relaxed phenotype after glucose limitation (relG) has been characterized further. Analysis of this mutant and of the downshift mechanism in B. subtilis has shown that the primary defect lies in glucose uptake.

Saccharomyces cerevisiae-secreted fusion proteins Pfs25 and Pfs28 elicit potent Plasmodium falciparum transmission-blocking antibodies in mice.

Transmission-blocking vaccines based on sexual-stage surface antigens of Plasmodium falciparum may assist in the control of this lethal form of human malaria. Two vaccine candidates, Pfs25 and Pfs28, were produced as single recombinant fusion proteins. The 39-kDa chimeric proteins, having a C-terminal His6 tag, were secreted by Saccharomyces cerevisiae, using the prepro-alpha-factor leader sequence. Pfs25-28 fusion proteins were significantly more potent than either Pfs25 or Pfs28 alone in eliciting antibodies in mice that blocked oocyst development in Anopheles freeborni mosquitoes: complete inhibition of oocyst development in the mosquito midgut was achieved with fewer vaccinations, at a lower dose, and for a longer duration than with either Pfs25 or Pfs28 alone. Increased antigen-specific immunoglobulin G titers and highly significant lymphoproliferative stimulation by Pfs28-containing antigens suggest the presence of an immunodominant helper T-cell epitope in the Pfs28 portion of the fusion proteins. This epitope may be responsible for the enhanced humoral response to both Pfs25 and Pfs28 antigens. Protein production of the fusion protein was improved 12-fold by converting Pfs28 codons to yeast-preferred codons (TBV28), using a modified ADH2 promoter and incorporating a (Glu-Ala)2 repeat after the Kex2 cleavage site.

Either one of the two yeast EF-1 alpha genes is required for cell viability.

Two genes, TEF1 and TEF2, encode the protein elongation factor EF-1 alpha in the yeast Saccharomyces cerevisiae. We have generated yeast haploid strains containing either TEF1 or TEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1 alpha genes are viable. In contrast, attempts to isolate a yeast haploid strain with both TEF1 and TEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.

Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate.

Gozar, M. M. G., Muratova, O., Keister, D. B., Kensil, C. R., Price, V. L., and Kaslow, D. C. 2001. Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate. Experimental Parasitology 97, 61-69. The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice. Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits. Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21. As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations. Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28. The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28.

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha.

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.

Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae.

Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications.