Mycobacterium recombinant vaccines.

Intracellular diseases are difficult to treat and constitute a major problem for modern medicine. In this type of diseases, a TH-1 immune response favors protection, while a TH-2 response is detrimental to the host. Current vaccines are using antigens to initiate an immune response regardless of its nature and its mechanism. New vaccines are designed to combine selected antigens with potent adjuvants to stimulate the appropriate pathway of the immune system and deliver a lasting protective immunity. The Mycobacterium recombinant vaccine system for treatment of intracellular diseases utilizes antigen delivery systems in the form of non pathogenic Mycobacterium strains, genetic transfer systems in the form of cloning and expression vectors, and related technologies to provide products containing non toxic immuno-regulating Mycobacterium adjuvants, non toxic immuno-stimulating exogenous antigens specific for a variety of diseases, and non toxic amounts of cytokines that boost the TH-1 pathway. The cloning and expression Mycobacterium vectors include both pAL5000-based extra-chromosomal and D29-based integrative vectors.

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products.

The anti-cancer drug taxol binds to beta-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC50 greater than 11.7 microM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 microM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding beta-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of beta-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of beta-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [3H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [3H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various beta-tubulin sequences showed that beta-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but beta-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and beta-tubulin.

Tumor necrosis factor activities and cancer therapy--a perspective.

Tumor necrosis factor (TNF) is a multifunctional cytokine which has excited and fascinated numerous investigators and commercial entities due to its promise as a therapeutic agent against cancer and as a target for drugs treating septic shock. TNF is a protein having cytotoxic, cytostatic, immunomodulatory as well as several other activities and is also involved in septic shock. This review covers the structure of TNF and its receptors, various in vitro activities and in vivo activities based on studies in animal model systems. The role of TNF as an anticancer therapeutic agent, based on various phase I and phase II clinical studies, has also been considered. The review concludes with several considerations for increasing the therapeutic utility of TNF in terms of targeting, toxicity and half-life.

Selection of secretory protein-encoding genes by fusion with PHO5 in Saccharomyces cerevisiae.

Secretory protein-encoding genes of Saccharomyces cerevisiae have been cloned by a novel procedure that is based on the functional selection of their fusions with acid phosphatase (APase) at the DNA level. DNA fragments that functionally replace the promoter and signal sequence-encoding regions of the PHO5 gene (encoding APase) have been obtained by positive selection from a pool of cloned random DNA fragments. Five unique DNA sequences containing the promoter, and encoding signal sequences have been isolated. We have also isolated the complete gene, SSP120, encoding one of these S. cerevisiae secretory proteins, SSP120. Gene disruption studies have shown that the SSP120 gene is not essential for viability and growth. The SSP120 amino acid (aa) sequence has 13.5% identity with the middle 88-250 aa residues of the chicken glycosylation site-binding protein. However, SSP120 disruption did not affect protein glycosylation in yeast. The present study provides an alternative approach for the isolation of genes encoding secretory proteins, in contrast to classical genetic approaches that require isolation of functionally defective mutations followed by gene isolation by functional complementation. The present procedure should contribute to our understanding of protein sorting by permitting the cloning of genes encoding proteins targeted to different organelles in the secretory pathway.

Expression and modulation by cytokines of the intercellular adhesion molecule-1 (ICAM-1) in human central nervous system tumor cell cultures.

The intercellular adhesion molecule (ICAM-1) has been shown to be important in interactions involving cells of the immune system and to be upregulated in a number of cell culture systems by cytokines, including immune interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In the present study, we have determined by fluorescence-activated cell sorter (FACS) analysis and the anti-ICAM-1 monoclonal antibody (MAb) CL203.4 the base-line expression of ICAM-1 and its modulation by recombinant IFN-beta, IFN-gamma and TNF-alpha in early passage (less than 15) human central nervous system (CNS) tumor-derived cell cultures. These cultures were established from various malignancies, including glioblastoma multiforme (GBM), astrocytoma (AST), ganglioglioma, medulloblastoma, meningioma and a pineal tumor. ICAM-1 expression was highest in the GBM- and AST-derived cell cultures and was lowest in the ganglioglioma and normal pineal cell cultures. Variable ICAM-1 expression was found, however, in tumors of the same histological group. In several cell cultures the variable expression observed by FACS was substantiated by the intensity of the molecular species immunoprecipitated by the anti-ICAM-1 MAb CL203.4 from these cells. All the cell cultures displayed variable but consistent increases in ICAM-1 expression following treatment with IFN-gamma or TNF-alpha. In general, the degree of increase in ICAM-1 expression was greatest in cultures exposed to TNF-alpha. Upregulation of ICAM-1 expression in an established glioblastoma multiforme cell line was of greater magnitude and more rapid following TNF-alpha treatment (within 2 to 3 hr) than exposure to IFN-gamma (by 24 hr). In several cultures, IFN-beta also increased ICAM-1 expression and enhanced the increase induced by TNF-alpha. The results of the present study indicate that variable expression of ICAM-1 is a common property of early passage cultures of CNS tumors and recombinant interferons and TNF-alpha can differentially upregulate ICAM-1 expression in these CNS tumor cell cultures.

Bacterial plasmid pBR322 sequences serve as upstream activating sequences in Saccharomyces cerevisiae.

The expression of acid phosphatase (APase) from PHO5 and MF alpha-PHO5 hybrid genes is regulated by inorganic phosphate and mating type locus respectively, as well as the PHO4 and MAT alpha 1 gene products respectively. When PHO5 and MF alpha-PHO5 hybrid genes were cloned in the BamHI site of the pBR322 sequence of the yeast shuttle vectors (YRp7 or YEp9T), in one orientation they were regulated normally but in the other orientation their expression was not regulated but expressed constitutively. The pBR322 sequences present upstream of the inserted genes are responsible for the constitutive expression. By replacing the PHO5 upstream activating sequences (UAS) element with pBR322 fragments, we have identified three pBR322 sequences, from nucleotides 376 to 650, 2068 to 2116 and 2136 to 2247, which were able to promote expression of APase. A comparison of these three pBR322 fragments revealed 5' ATCGCGCGAG 3' and 5' CGGTGATGNCGG 3' to be the common sequences likely to act as UASs in Saccharomyces cerevisiae. By using synthetic oligonucleotides, it was found that both sequences are required for maximum expression of APase activity.

Tumor necrosis factor analogs: identification of functional domains.

Truncated tumor necrosis factor analogs were produced by making in vitro deletions of the coding sequence of the human TNF gene. One of these analogs, TNF-desA7, lacked seven amino terminal residues of the mature TNF protein and the other two analogs, TNF-desC7 and TNF-desC2, had deletions of seven and two carboxy terminal amino acids, respectively. While the deletion of the first seven amino acid residues did not affect the biological activity of the protein, the deletions of carboxy terminal residues in both analogs resulted in the complete loss of biological activity. A direct correlation of the biological activity of the TNF protein and its binding to neutralizing monoclonal antibodies was observed. The carboxy terminal-deleted TNF analogs, with no biological activity, did not bind to neutralizing monoclonal antibodies while the amino terminal-deleted analog, which retained complete biological activity, did bind. These results indicate that the carboxy terminal amino acids of the TNF protein are essential for TNF biological activity and are part of an epitope recognized by neutralizing monoclonal antibodies.

Expression of c-myc and c-Ha-ras oncogenes in human lymphoblastoid cells (Namalva).

We have studied the expression of c-myc and c-Ha-ras oncogenes in this cell line. Daudi lymphoblastoid cells and normal human leukocytes served as a positive and a negative control, respectively. The Northern blot analysis using a c-myc probe revealed a 2.7 kb transcript and two larger transcripts greater than 23 kb. In the Northern blot hybridization using a c-Ha-ras probe, two transcripts with sizes of 1.8 and 6.1 kb were observed. The affinity of the Namalva RNA hybridization to c-myc probe was about 10-fold lower than that in Daudi RNA, whereas no difference in the c-Ha-ras hybridization was observed between the two cell lines. These data indicate that c-myc and c-Ha-ras are expressed in Namalva cells. It is noteworthy to consider that the difference in oncogene expression between Namalva and Daudi cells might be due to the difference in interferon properties between the two cell lines.

Human cytokines, tumor necrosis factor, and interferons: gene cloning, animal studies, and clinical trials.

Presented is a comprehensive program designed to isolate human cytokine genes and investigate their relative induction, and to analyze cytokine activities in cell culture, animal tumor models, and human clinical trials. Human cytokine cDNAs have been isolated from a cDNA library made from normal human peripheral blood leukocytes (PBLs) treated with Sendai virus and the relative induction of tumor necrosis factor (TNF), alpha and gamma interferons (IFN-alpha, IFN-gamma), and interleukin-1 beta IL-1 beta) genes has been analyzed. In the Sendai virus-induced PBL system, IL-1 beta mRNA was shown to be approximately twofold higher than TNF or IFN-alpha mRNA whereas IFN-gamma mRNA was 50-100-fold lower than TNF or IFN-alpha mRNA. The cytotoxic activity of TNF was analyzed on several cell lines and IFN-alpha and IFN-gamma were shown to potentiate TNF cytotoxicity about 2-200-fold depending on cell lines. The LD50 for recombinant TNF in BALB/c mice was determined to be 6 X 10(7) U/kg and the therapeutic dose of recombinant TNF in sarcoma 180 bearing BALB/c mice was 3 X 10(5) U/kg, indicating a wide therapetic index. Phase I clinical trials of recombinant TNF given I.V. indicated a tolerated dose of 150,000 U/kg with biphasic half-life (T-1/2) of 2 and 31 min following TNF injection. Phase II trials of TNF and trials of TNF combined with IFN-alpha are in progress. These studies indicate that cytokines such as TNF and IFN-alpha are subject to similar induction systems, potentiate each other's activities, and can be tolerated at specific doses for potential therapeutic use.

Analysis of alpha-factor secretion signals by fusing with acid phosphatase of yeast.

In yeast, Saccharomyces cerevisiae, the PHO5 gene encodes the repressible acid phosphatase (APase) whose activity can be easily monitored by either the staining of colonies or by colorimetric assay. Therefore, gene fusions to PHO5 provide a convenient system for structural and functional analysis of yeast genes. We have constructed fusions of the PHO5 gene with a MF alpha 1 gene of yeast to delineate the secretion signal(s) in the alpha-factor leader peptide. Gene fusion between MF alpha 1 and PHO5 codes for a hybrid protein in which the alpha-factor leader peptide of 89 amino acids (aa) directed the export of APase, a periplasmic protein, into the medium. Since the hybrid gene is transcribed from the alpha-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation. Further analyses of another MF alpha 1-PHO5 fusion showed that only the first 22 aa of the 89-aa alpha-factor leader peptide contained sufficient information for the secretion of APase into the medium. This shows that, in addition to the analysis of gene regulation, PHO5 fusions can be used to study signals involved in the proper localization of proteins.

Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes.

Sendai virus induces human peripheral blood leukocytes to produce high levels of tumor necrosis factor (TNF) mRNA. TNF mRNA can represent as much as 0.6% of the total mRNA. Kinetic studies indicate that the level of TNF mRNA peaks about 2 hours before that of IFN-alpha mRNA produced in the same system. Although the peak levels of TNF and IFN-alpha mRNA were similar, TNF in the culture supernatants was at a 200 fold lower level than IFN-alpha. Cloning and sequence analysis of TNF cDNA isolated from peripheral blood leukocytes RNA showed that normal human cells in response to Sendai virus produce TNF identical to that previously isolated and cloned from tumor-derived cell lines. A bacterial expression system was used to produce the cloned TNF at a maximum level of 2 X 10(6) units per ml of culture.

Oncogene expression in human hepatoma cells PLC/PRF/5.

The expression of 7 cellular oncogenes in a human hepatoma cell line PLC/PRF/5 was studied using Northern blot analyses. Among the oncogenes tested, c-abl, c-fes, c-fms, c-myc, c-Ha-ras and c-sis were expressed. The oncogene c-Ki-ras was not expressed. The length of the mRNAs expressed was almost consistent with published data. Compared to the oncogene expression in Daudi lymphoma cells, the same kind of oncogenes were expressed in PLC/PRF/5 cells, but the intensity of the signal in each oncogene expression was stronger in Daudi cells than in PLC/PRF/5 cells. Considering the cellular localization and the function of each oncogene, the oncogene survey in hepatoma cells broadens the knowledge of hepatocarcinogenesis and the character of human hepatoma cells.

Cloning and sequencing of a human 18S ribosomal RNA gene.

A clone containing an 18S ribosomal RNA (rRNA) gene has been isolated from a human genomic library constructed in lambda Charon 4A. This gene was sequenced and found to be 1868 bp long. The sequence divergencies in the human 18S rRNA gene and the previously sequenced mouse and rat genes are found in one G + C-rich region of 110 bp located in the 5' domain of the molecule. Except for this variable region, extensive homology exists among these three mammalian genes. Overall, the human 18S rRNA gene is 98.8% homologous with those of rat and mouse.

Pseudogene IFN-alpha L: removal of the stop codon in the signal sequence permits expression of active human interferon.

Biologically active interferon (10(6)-10(7) units/liter) was produced in Escherichia coli from modified human alpha interferon (IFN-alpha) pseudogene L. IFN-alpha pseudogene L has a stop codon in the signal peptide coding region. The region that contains the stop codon was replaced with the corresponding region of another human IFN-alpha gene, WA, that does not have a stop codon and was previously engineered for expression by fusion to the M13mp11 lac promoter. The interferon L fusion product was induced with IPTG after infecting E. coli JM103 with the M13 bacteriophage that contained the modified human IFN-alpha pseudogene L. Hence, the IFN-alpha L mature interferon coding sequence, which is not identical to any other alpha-interferon gene, has been conserved for active interferon coding information.

Human genomic library screened with 17-base oligonucleotide probes yields a novel interferon gene.

A method is presented that has permitted a human genomic library to be screened for low-copy genes using 17-base synthetic oligonucleotides as probes. Parallel screening with two different 17-base probes permitted the unambiguous identification of clones containing interferon-alpha (IFN-alpha) genes. The isolated human IFN-alpha genes were sequenced, and one appears to be IFN-alpha L; the other is one not previously described, which we have designated IFN-alpha WA. The IFN-alpha WA sequence differs from those of IFN-alpha genes A-L at approximately equal to 10% of the positions and is most similar to IFN-alpha C, -alpha F, and -alpha H. IFN-alpha WA has been found to encode amino acids that differ from those conserved at each of five positions in all previously reported IFN-alpha species. The IFN-alpha WA gene codes for an active interferon, which has been expressed in Escherichia coli using an M13-lacZ fusion as an expression vector. About 5 X 10(6) units of IFN-alpha WA were obtained per liter of bacterial culture. The described screening procedure using short probes should permit the isolation of genes for which sequence information is available from animal or plant genomic libraries.

The complete nucleotide sequence of the rat 18S ribosomal RNA gene and comparison with the respective yeast and frog genes.

The complete nucleotide sequence of the rat 18S ribosomal RNA gene has been determined. A comparison of the rat 18S ribosomal RNA gene sequence with the known sequences of yeast and frog revealed three conserved (stable) regions, two unstable regions, and three large inserts. (A,T) leads to (G,C) changes were more frequent than (G,C) leads to (A,T) changes for three comparisons (yeast leads to frog, frog leads to rat, and yeast leads to rat). GC pairs were inserted preferentially over AT pairs for the same three comparisons. These two factors contribute to the progressively higher GC content of 18S ribosomal RNA of yeast, frog, and rat.

Nucleotide sequence of the 5'-terminal region of rat 18S ribosomal DNA.

The 5'-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gee and 10 bp of the adjoining transcribed spacer have been sequenced. Previously sequenced 10 large oligonucleotides of rat 18S RNA were located in this region. This mammalian sequence has been compared with the known sequences of yeast and frog 18S rDNA's. The analysis indicates that 534 bp of the 597 bp (89%) are conserved between rat and frog sequences but only 75% of the nucleotides are conserved between rat and yeast in this region. Two large and two small sections have been identified where insertions have been introduced during evolution. Of these 58 bp long inserted sections of the rat rDNA sequence, 50 bp (86%) were G-C base-pairs.

Nucleotide sequence of the 3'-terminal region of rat 18S ribosomal DNA.

The 3'-terminal 230 base-pairs (bp) of the gene for 18S rRNA and 40 bp of the adjoining spacer have been sequenced for the Sprague-Dawley rat. This mammalian sequence has been compared with the known sequences of yeast, fruit fly, silkworm, and frog. This study has shown that the nucleotide-sequence differences between rat and frog are the smallest among and longer species, probably reflecting their evolutionary closeness and longer maturation time compared to the others. There is little similarity in the nucleotide sequences of the transcribed spacer regions of the five species compared.

Analysis of yeast ilv 1 CIS control and domain mutants.

We have identified a cis control region specific for the ilv 1 gene of Saccharomyces cerevisiae. Mutants designated ilv 1-OPc map in this control region located between arg 6 and ilv 1 and result in increased basal levels and constitutive synthesis of the ilv 1 gene products. Furthermore, ilv 1 mutants have been isolated in three different structural domains indicating that the ilv 1 gene may contain a functional intervening sequence specific for one of the two gene products.