Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

OBJECTIVE: To develop a slow-release carboplatin formulation for intratumoral administration to cats. DESIGN: Preliminary study to analyze pharmacokinetic effects of purified sesame oil in the carboplatin formulation for intratumoral administration, and a second study to evaluate the efficacy and toxicosis of intratumoral administration of carboplatin in purified sesame oil. ANIMALS: 23 cats with squamous cell carcinomas of the nasal plane. PROCEDURE: Eight cats with advanced-stage tumors were submitted to intratumoral administration of 100 mg of carboplatin/m2 of body surface area, with or without purified sesame oil, using a two-period, cross-over design. Fifteen additional cats were treated by intratumoral administration of carboplatin in purified sesame oil. Four weekly intratumoral chemotherapy injections of carboplatin in purified sesame oil at a dosage of 1.5 mg/cm3 of tissue were given. RESULTS: Purified sesame oil in the formulation significantly reduced systemic exposure to carboplatin and drug leakage from the sites of injection. Cumulative effects of repeated intratumoral administrations on plasma concentrations of carboplatin were not observed. Systemic toxicosis was not observed, and local toxicosis was minimal. Healing of ulcerated lesions was not compromised. Rates of complete clinical tumor clearance and complete response were 67 and 73.3%, respectively. Product-limit estimates of mean progression-free survival times was 16 +/- 3.3 months. The 1-year progression-free survival rate was 55.1 +/- 13%. Local recurrence was observed in 7 cats; 4 had marginal tumor recurrence, and 3 had in-field and marginal tumor recurrence. CONCLUSIONS: Intratumoral carboplatin chemotherapy is safe and effective for cats with squamous cell carcinoma of the nasal plane. Future studies to improve treatment efficacy could include evaluation of increased dose-intensity as well as combination of this modality with radiotherapy. CLINICAL RELEVANCE: Intratumoral administration of carboplatin in a water-sesame-oil emulsion was found to be a practical and effective new treatment for facial squamous cell carcinomas in cats.     

Detection of maltose fermentation genes in the baking yeast strains of Saccharomyces cerevisiae

The presence of any one of the five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 and MAL6) confers the ability to ferment maltose on the yeast Saccharomyces cerevisiae. Each locus is composed of three genes encoding maltose permease, alpha-glucosidase and MAL activator. Chromosomal DNA of seven representative baking strains has been separated by pulse-field gel electrophoresis and probed with three genes in MAL6 locus. The DNA bands to which all of the three MAL-derived probes simultaneously hybridized were chromosome VII carrying MAL1 in all of the strains tested, chromosome XI carrying MAL4 in six strains, chromosome III carrying MAL2 in three strains and chromosomes II and VIII carrying MAL3 and MAL6, respectively, in the one strain. The number of MAL loci in baking strains was comparable to those of brewing strains.     

A family of hexosephosphate mutases in Saccharomyces cerevisiae

The Saccharomyces cerevisiae PGM1 and PGM2 genes encoding two phosphoglucomutase isoenzymes have been isolated and sequenced. The derived protein sequences are closely related to one another and show distinct sequence similarities to the human and rabbit phosphoglucomutases, especially in the region supposed to constitute the active site. PGM1 and PGM2 are located on chromosomes XI and XIII, respectively, just upstream of the known genes YPK1 and YKR2 coding for a pair of closely related putative protein kinases. These observations suggest that an extended region of DNA arose by the process of gene duplication. Cells deleted for both, PGM1 and PGM2, could not grow on galactose. No residual phosphoglucomutase activity could be measured in crude extracts or in permeabilized cells of pgm1/2 double mutants. Unexpectedly, growth with glucose was not impaired and the mutant cells were still able to accumulate trehalose and glycogen, although at a reduced level. Two further genes could be isolated and characterized which when over-expressed on a multi-copy plasmid could restore growth on galactose of the pgm1/2 double deletion mutant. Multi-copy complementation was due to a sharply increased level of phosphoglucomutase activity. Partial sequencing and characterization of the two genes revealed one of them to be SEC53 encoding phosphomannomutase. No extended sequence similarities could be found in the databases for the second gene. However, part of the derived amino acid sequence contained a region of high similarity to the active-site consensus sequence of hexosephosphate mutases from different organism. Further investigations suggest that a complex network of mutases exist in yeast which interact and can partially substitute for each other.     

SN-FORTH/MSX: a state notation system for controlling behavioral experiments with a low-priced computer

A software system, SN-FORTH/MSX, has been developed to control behavioral experiments with a low-priced personal computer, MSX. Designed on the model of state notation, SN-FORTH/MSX enables researchers to program easily a wide variety of behavioral procedures including complex processes that are hard to describe with conventional programming languages such as BASIC and C. Since the real-time monitor of this system has a capability of parallel processing of multiple state-sets, several independent experiments can be simultaneously conducted with a single computer. Users can observe and modify the working of real-time monitor in each instant with a set of run-time commands supplied in the system.     

Improvement of nitrogen assimilation and fermentation kinetics under enological conditions by derepression of alternative nitrogen-assimilatory pathways in an industrial Saccharomyces cerevisiae strain

Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele of ure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO2 production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.     

The anatomy of a hypoxic operator in Saccharomyces cerevisiae

Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes.     

Menadione toxicity in Saccharomyces cerevisiae cells: activation by conjugation with glutathione

Menadione (2-methyl-1,4-naphthoquinone) has been used extensively as an oxidant stressor at the cellular level. However, the mechanism of cytotoxicity of this compound still remains controversial. This study deals with the role of intracellular glutathione in the resistance of the yeast Saccharomyces cerevisiae to menadione. Incubation with 0.5 mM menadione resulted in a decrease of total glutathione concentration in yeast cells, intracellular formation of menadione S-glutathione conjugate and export of the conjugate from cells. GSH-deficient mutants showed lower stimulation of superoxide and hydrogen peroxide production upon exposure to menadione and were more resistant to menadione than wild-type isogenic strains. These results indicate that in yeast cells the formation of S-glutathione conjugate is a major pathway of menadione metabolism and that this reaction leads to redox activation of menadione but permits its removal from the cells.     

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site

Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli. By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues. This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg. The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, [D253K]HCPB. Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn. These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer.     

Glucose-induced sequential processing of a glycosyl-phosphatidylinositol-anchored ectoprotein in Saccharomyces cerevisiae

Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.     

Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose

What is neuroinformatics? What is the Human Brain Project? Why should you care? Supported by a consortium of US funding agencies, the Human Brain Project aims to bring to the analysis of brain function the same advantages of Internet-accessible databases and database tools that have been crucial to the development of molecular biology and the Human Genome Project. The much greater complexity of neural data, however, makes this a far more challenging task. As a pilot project in this new initiative, we review some of the progress that has been made and indicate some of the problems, challenges and opportunities that lie ahead.     

Purification and characterization of a DNA helicase from Saccharomyces cerevisiae

A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.     

A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.     

A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism

Many secreted signaling molecules are synthesized as precursors that undergo multiple maturation steps to generate their mature forms. The Saccharomyces cerevisiae mating pheromone a-factor is a C-terminally isoprenylated and carboxylmethylated dodecapeptide that is initially synthesized as a larger precursor containing 36 or 38 amino acids. We have previously shown that the maturation of a-factor occurs by an ordered biogenesis pathway involving 1) three C-terminal modification steps, 2) two N-terminal proteolytic processing events, and 3) a nonclassical export mechanism mediated by the ATP-binding-cassette (ABC) transporter Ste6p. In the present study, we demonstrate that an unexpected and abundant a-factor-related peptide (AFRP) exists in the culture fluid of MATa cells and that its biogenesis is integrally related to that of mature a-factor itself. We show by purification followed by mass spectrometry that AFRP corresponds to the C-terminal 7 amino acids (VFWDPAC) of mature a-factor (YIIKGVFWDPAC), including both the farnesyl- and carboxylmethylcysteine modifications. The formation and export of AFRP displays three striking features. First, we show that AFRP is produced intracellularly and that mutants (ste24 and axl1) that cannot produce mature a-factor due to an N-terminal processing defect are nevertheless normal for AFRP production. Thus, AFRP is not derived from mature a-factor but, instead, from the P1 form of the a-factor precursor. Second, fusion constructs with foreign amino acids substituted for authentic a-factor residues still yield AFRP-sized molecules; however, the composition of these corresponds to the altered residues instead of to AFRP residues. Thus, AFRP may be generated by a sequence-dependent but length-specific proteolytic activity. Third, a-factor and AFRP use distinct cellular machinery for their secretion. Whereas a-factor export is Ste6p-dependent, AFRP is secreted normally even in a ste6 deletion mutant. Thus, AFRP may exit the cell by another ATP-binding-cassette transporter, a different type of transporter altogether, or possibly by diffusion. Taken together, these studies indicate that the biogenesis of AFRP involves novel mechanisms and machinery, distinct from those used to generate mature a-factor. Because AFRP neither stimulates nor inhibits mating or a-factor halo activity, its function remains an intriguing question.     

Comparative characterization of the fermentation pathway of Saccharomyces cerevisiae using biochemical systems theory and metabolic control analysis: model definition and nomenclature

Mathematical tools that involve the determination of systemic responses to small changes in metabolites or enzymes have demonstrated their utility for analyzing metabolic pathways. The different methodologies based on these ideas allow for modeling and analyzing biochemical pathways focusing on the coordinate behavior of the whole system. However, one must become familiar with the difference in nomenclature and methodology to relate the models and results obtained by applying these techniques and to appreciate their potential for answering fundamental questions about biochemical systems. In the following three papers we show how this can be facilitated by comparing the nomenclature, methodology, and results of the two leading techniques in this area, metabolic control analysis and biochemical systems theory, using a model of the fermentation pathway in Saccharomyces cerevisiae as a reference system. In the present paper we review the nomenclature, technical concepts, and related experimental measurements while creating a practical dictionary for the reference system that makes the relatedness of the two approaches more apparent. In the second paper, subtitled Steady-State Analysis, we show that both approaches give the same picture for many systemic responses of the reference system. In the third paper of this series, subtitled Model Validation and Dynamic Behavior, we show that the quality of the model can be assessed by studying the sensitivity to changes in the system parameters. We hope to illustrate the usefulness of these tools in providing an interpretation of the experimental measurements in a specific metabolic pathway.     

A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae

Aerobically growing wild-type strains of Saccharomyces cerevisiae are unable to take exogenously supplied sterols from media. This aerobic sterol exclusion is vitiated under anaerobic conditions, in heme-deficient strains, and under some conditions of impaired sterol synthesis. Mutants which can take up sterols aerobically in heme-competent cells have been selected. One of these mutations, designated upc2-1, gives a pleiotropic phenotype in characteristics as diverse as aerobic accumulation of sterols, total lipid storage, sensitivity to metabolic inhibitors, response to altered sterol structures, and cation requirements. During experiments designed to ascertain the effects of various cations on yeast with sterol alterations, it was observed that upc2-1 was hypersensitive to Ca2+. Using resistance to Ca2+ as a screening vehicle, we cloned UPC2 and showed that it is YDR213W, an open reading frame on chromosome IV. This belongs to a fungal regulatory family containing the Zn(II)2Cys6 binuclear cluster DNA binding domain. The single guanine-to-adenine transition in upc2-1 gives a predicted amino acid change from glycine to aspartic acid. The regulatory defect explains the semidominance and pleiotropic effects of upc2-1.