Arthur P. Bollon

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.

Acremonium sp.—a leucinostatin A producing endophyte of European yew (Taxus baccata)

Abstract Acremonium sp. occurs as an endophyte in European yew ( Taxus baccata ). It produces a series of peptide antifungal-anticancer agents known as the leucinostatins. Leucinostatin A is especially active against the oomycetous—plant pathogenic fungus— Pythium ultimum with an effective 1 day 50% inhibitory concentration of μ mol. Leucinostatin A also possesses activity against certain human cancer cell lines, for instance, its IC 50 value is 2.3 nM for breast cancer cell line BT-20 contrasted with 640 nM for a normal mammary cell line. Leucinostatin A can be effectively prepared radiolabeled via the administration of [ 14 C]leucine or [ 14 C]pyruvate to standing cultures of Acremonium sp. The data point to a biological role of antifungal agents being produced by endophytic fungi as a means to allow for their survival. Also, since antifungal agents such as taxol and leucinostatin A are produced by some endophytic fungi, oomycetes such as P. ultimum may serve as initial screening tools for anticancer agents.

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

Abstract 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 MFα1 gene of yeast to delineate the secretion signal(s) in the α-factor leader peptide. Gene fusion between MFα1 and PHO5 codes for a hybrid protein in which the α-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 α-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation. Further analyses of another MFαl-PHO5 fusion showed that only the first 22 as of the 89-aa α-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.

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

Abstract The anti-cancer drug taxol binds to β-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 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-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 β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-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 β-tubulin.

Regulation of the ilv 1 multifunctional gene in Saccharomyces cerevisiae.

Summary1.The ilv1 gene in S. cerevisiae codes for a regulatory protein involved in depression of the ilv 2 and ilv 3 genes as well as a biosynthetic enzyme, threonine deaminase.2.The ilv 1 gene does not autogenously regulate its catalytic product threonine deaninase.3.Regulation of the ilv 2 and ilv 3 gene products involve different aporepressors than regulation of the ilv 1 gene product.4.The ilv 1 multifunctional gene in S. cerevisiae may be a duplication and fusion of a bacterial like ilv 1 gene where ilv 1 catalytic and regulatory functions have been differentially conserved.

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.

Pseudogene IFN-αL: 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.

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

SummaryThe 5′-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gene 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 rDNAs. 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.

[53] A procedure for isolation of alpha interferon genes with short oligonucleotide probes

Publisher Summary The chapter describes a procedure for the isolation of alpha interferon genes with short oligonucleotide probes. A method has been developed, which permitted the isolation of several human alpha interferon (IFN-α) genes from a human genomic library in Charon 4A bacteriophages with 17-base synthetic oligonucleotide probes. Previously, human IFN-α genes are isolated from complementary DNA (cDNA) libraries or genomic libraries with cDNA probes. The use of select, short, synthetic probes permits the isolation of the subsets of genes within a gene family represented in a genomic library. The screening procedure with 17-base probes described in the chapter is useful for the isolation of most genes from a human genomic library, although some conditions will vary according to the sequence of the probes. Procedure yielded three IFN-α genes, which had perfect homology with the two probes utilized. Even genes containing introns could be isolated from a genomic library with short and mixed probes provided the intron does not split the sequences of the probes to a size unable to maintain a stable hybrid. Changes at positions 42 and 133 result in the replacement of acidic residues with hydrophobic residues.