Assimilation of grape phytosterols by Saccharomyces cerevisiae and their impact on enological fermentations.

Although yeasts are known to be able to incorporate a wide variety of exogenous sterols under strict anaerobiosis, no data are available on the assimilation of grapevine phytosterols under enological conditions and the eventual impact on fermentation kinetics. We used therefore a mixture of pure phytosterols, in a proportion representative of the different grape skins phytosterols, to supplement a synthetic fermentation medium simulating a grape must. Under anaerobiosis, normal biomass formation was achieved with 5 mg phytosterols l(-1). Similar results were obtained in comparison with the observed maximal fermentation rates. These results clearly indicated that grape phytosterols may efficiently act as a substitute for ergosterol in the yeast membrane for promoting yeast growth and initial fermentative activity. Analysis of total yeast sterols indicated that phytosterols are accumulated without further modification, mainly in their esterified form. However, all the fermentations performed with synthetic media supplemented with phytosterols led to stuck fermentations, linked to a correlative strong decrease in cell viability during the stationary phase. Therefore, grape phytosterols are easily incorporated by yeast cells under enological conditions for promoting initial growth and fermentative activity, but rapidly perturb the yeast membrane properties by being the predominant sterols.

Caelyx/Doxil for the treatment of metastatic ovarian and breast cancer.

Caelyx/Doxil is a novel pegylated liposomal formulation of the first-generation anthracycline, doxorubicin. The pharmacokinetics of this polyethylene-glycol-coated liposome are characterized by a reduced volume of distribution, a long intravascular circulating half-life and slow plasma clearance compared with free doxorubicin. This, coupled with a small vesicular size, uniquely promotes the localization of Caelyx/Doxil at tumor sites and explains its altered toxicity profile. The FDA and EMEA have approved its use for the treatment of AIDS-related Kaposi's sarcoma and, more recently, for recurrent epithelial ovarian cancer (EOC). Numerous investigations have focused on its use in the treatment of metastatic breast cancer, as well as recurrent squamous cell cervical carcinoma, soft tissue sarcoma, squamous head and neck cancers, prostate cancers and malignant gliomas. Ongoing clinical studies of combination regimens incorporating Caelyx/Doxil will further clarify its role in the treatment of advanced solid tumors.

Characterization, heterologous expression and functional analysis of mevalonate diphosphate decarboxylase gene (MVD) of Candida albicans.

Mevalonate diphosphate decarboxylase (MVD) catalyzes the conversion of mevalonate diphosphate to isopentenyl diphosphate, a key building block for a large family of functionally important biomolecules. We have cloned the gene encoding MVD from Candida albicans, and report the first characterization of such a gene from an opportunistic fungal pathogen. Sequence analysis revealed that the MVD comprises 362 amino acid residues with a predicted molecular weight of 39.5 kDa, sharing minimal identity with the human analogue. Analysis of the genomic sequence indicated that the coding frame is interrupted by a small intron of 51 bp. Southern analysis of genomic DNA demonstrated that MVD is a single-copy gene. Furthermore, Southern analysis of electrophoretic karyotypes of C. albicans obtained by pulsed-field gel electrophoresis showed that MVD is located on chromosome 1. Northern analysis revealed that the level of MVD expression is affected by (1) the carbon source in the growth medium, (2) the growth phase, and (3) the growth form of the fungus (yeast-like or hyphal). To demonstrate the biological function of C. albicans MVD, complementation experiments were carried out with an S. cerevisiae strain (erg19(ts)) that is temperature-sensitive for MVD activity. A single copy of the C. albicans MVD gene, under the control of the NOP1 promoter, was able fully to complement the erg19(ts) phenotype, and expression of the epitope-tagged C. albicans MVD was detectable by Western analysis. Furthermore, the low degree of sequence identity between C. albicans MVD and its human analogue raises the possibility that fungal-specific inhibitors can be developed for the enzyme. Thus, C. albicans MVD appears to be an interesting candidate that could be targeted for the development of anti-fungal agents.

SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae.

SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn[II]2Cys-6 family. It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells. A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction. Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters. Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene. We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)-Tup1p co-repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p. Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p. In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.

SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

Isolation and amino acid sequence of a peptide with vitellogenesis inhibiting activity from the terrestrial isopod Armadillidium vulgare (Crustacea).

The complete amino acid sequence of a neuropeptide was established using gas-phase microsequencing, mass spectrometry, and reverse transcription-polymerase chain reaction. This peptide, stored in the sinus gland of the terrestrial isopod Armadillidium vulgare, inhibited vitellogenin synthesis by the fat tissue and inhibited the onset of secondary vitellogenesis when tested in homologous bioassays. This peptide, named Arv-VIH, has 83 amino acid residues and a molecular mass of 9485 Da. Relationships with other related peptides are presented.

The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (erg19p) forms homodimers in vivo, and a single substitution in a structurally conserved region impairs dimerization.

The wild-type ERG19 gene of the yeast Saccharomyces cerevisiae encoding mevalonate diphosphate decarboxylase (MVD) and the mutated recessive erg19-34 allele leading to a decrease of sterol production and to a thermosensitive phenotype have been characterized [2]. The mutated erg19-34 allele bears a single amino acid leucine 79-to-proline (L79P) substitution. It was shown that this mutation does not affect the level of production of the enzyme. We performed a two-hybrid assay to show that the yeast Saccharomyces cerevisiae MVD forms homodimers in vivo and that the single point mutation drastically impairs the oligomerization of the protein, thereby explaining the deficiency of MVD activity observed in the temperature-sensitive strain.

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase.

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.

Molecular cloning, expression analysis, and chromosomal localization of human syntaxin 8 (STX8).

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations.

Nucleolar KKE/D repeat proteins Nop56p and Nop58p interact with Nop1p and are required for ribosome biogenesis.

Different point mutations in the nucleolar protein fibrillarin (Nop1p in Saccharomyces cerevisiae) can inhibit different steps in ribosome synthesis. A screen for mutations that are synthetically lethal (sl) with the nop1-5 allele, which inhibits pre-rRNA processing, identified NOP56. An independent sl mutation screen with nop1-3, which inhibits pre-rRNA methylation, identified a mutation in NOP58. Strikingly, Nop56p and Nop58p are highly homologous (45% identity). Both proteins were found to be essential and localized to the nucleolus. A temperature-sensitive lethal mutant allele, nop56-2, inhibited many steps in pre-rRNA processing, particularly on the pathway of 25S/5.8S rRNA synthesis, and led to defects in 60S subunit assembly. Epitope-tagged constructs show that both Nop56p and Nop58p are associated with Noplp in complexes, Nop56p and Nop1p exhibiting a stoichiometric association. These physical interactions presumably underlie the observed sl phenotypes. Well-conserved homologs are present in a range of organisms, including humans (52% identity between human hNop56p and yeast Nop56p), suggesting that these complexes have been conserved in evolution.

The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity.

The mevalonate diphosphate decarboxylase is an enzyme which converts mevalonate diphosphate to isopentenyl diphosphate, the building block of isoprenoids. We used the Saccharomyces cerevisiae temperature-sensitive mutant defective for mevalonate diphosphate decarboxylase previously described (C. Chambon, V. Ladeveve, M. Servouse, L. Blanchard, C. Javelot, B. Vladescu, and F. Karst, Lipids 26:633-636, 1991) to characterize the mutated allele. We showed that a single change in a conserved amino acid accounts for the temperature-sensitive phenotype of the mutant. Complementation experiments were done both in the erg19-mutated background and in a strain in which the ERG19 gene, which was shown to be an essential gene for yeast, was disrupted. Epitope tagging of the wild-type mevalonate diphosphate decarboxylase allowed us to isolate the enzyme in an active form by a versatile one-step immunoprecipitation procedure. Furthermore, during the course of this study, we observed that a high level of expression of the wild-type ERG19 gene led to a lower sterol steady-state accumulation compared to that of a wild-type strain, suggesting that this enzyme may be a key enzyme in mevalonate pathway regulation.

The astrocyte mitogen, tumor necrosis factor-alpha, inhibits the proliferative effect of more potent adult human astrocyte mitogens, gamma-interferon and activated T-lymphocyte supernatants.

The proliferative response of adult human astrocytes to tumor necrosis factor-alpha (TNF-alpha) was examined. Applied alone, TNF-alpha was dependent on the content of serum in the feeding medium, being mitogenic only in conditions of over 15% serum in medium. In accordance with previous results, supernatants from activated human CD8+ T-lymphocytes (CD8 SN) and recombinant human interferon-gamma (IFN-gamma) enhanced proliferation of adult human astrocytes in 5% serum-containing medium. Simultaneous administration of TNF-alpha (10-1000 units), however, abrogated the mitogenic effects of either CD8 SN or IFN-gamma; the inhibitory effect of TNF-alpha was lost if applied 2 days following IFN-gamma treatment. These studies show that while TNF-alpha is an astrocyte mitogen under selected conditions, it inhibits proliferation induced by other mitogens. In this manner, TNF-alpha may be important in regulating the proliferative response of astrocytes during reactive astrogliosis in vivo.

Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification.

The nucleolar protein fibrillarin (encoded by the NOP1 gene in yeast), is required for many post-transcriptional steps in yeast ribosome synthesis. A screen for mutations showing synthetic lethality with a temperature sensitive nop1-5 allele led to the identification of the NOP77 gene. NOP77 is essential for viability and encodes a nucleolar protein with a predicted molecular weight of 77 kDa. Depletion of NOP77p impairs both the processing and methylation of the pre-rRNA. The processing defect is greatest for the pathway leading to 25S rRNA synthesis, and is distinctly different from that observed for mutations in other nucleolar components. NOP77p contains three canonical RNA recognition motifs (RRMs), suggesting that it is an RNA binding protein. The NOP77 allele which complements the synthetic lethal nop1 strains has an alanine at position 308, predicted to lie in helix alpha 1 of RRM3, whereas the non-complementing nop77-1 allele contains a proline at the corresponding position. We propose that NOP77p mediates specific interactions between NOP1p and the pre-rRNA.

Reactive astrogliosis in the neonatal mouse brain and its modulation by cytokines.

Reactive astrogliosis is a characteristic response of astrocytes to inflammation and trauma of the adult CNS. To assess the hypothesis that cytokines from inflammatory mononuclear cells that accumulate around lesion sites have a role in modulating astrogliosis, this study sought to take advantage of the neonatal system in which astrogliosis is reported to be minimal following injury and in which the immune system is relatively immature compared to adult animals. A nitrocellulose membrane implant into the cortex of postnatal day 3 mice resulted in a tremendous astrogliotic response 4 d later, as measured by glial fibrillary acidic protein (GFAP) immunoreactivity and GFAP content. In contrast, a neonatal stab wound produced limited astroglial response when compared to the adult stab wound. Utilizing the neonatal stab wound model, cytokines were microinjected into the wound site at the time of injury. All cytokines tested (gamma-IFN, IL-1, IL-2, IL-6, TNF-alpha, and M-CSF) resulted in a significantly increased astrogliosis. The specificity of the cytokine response was demonstrated by the inability of human gamma-IFN, but not mouse gamma-IFN, in enhancing neonatal mouse astrogliosis, in accordance with reports that the interaction of gamma-IFN with its receptor occurs in a species-specific manner. We conclude that neonatal astrocytes can become reactive if an adequate injury stimulus is presented, and that the release of immunoregulatory cytokines by cells around lesion sites may be a mechanism that contributes to the production of gliosis.

Cloning of an Aspergillus niger invertase gene by expression in Trichoderma reesei.

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.

Purification and characterisation of an Aspergillus niger invertase and its DNA sequence.

A secreted invertase was purified 23-fold by ultrafiltration, ion-exchange, and gel filtration chromatography from the culture supernatant of 18 h sucrose-grown cultures of Aspergillus niger. The purified enzyme hydrolysed sucrose and raffinose but there was no detectable hydrolysis of inulin, melezitose or PNPG. Invertase activity was optimal at pH 5.5 and 50 degrees C. The molecular mass of reduced invertase was 115 kDa, as determined by SDS gel electrophoresis. The native molecular weight of between 225 kDa and 250 kDa, estimated by electrophoresis under non-denaturing conditions, suggests that the protein is a dimer of identical subunits. The suc1 gene encoding this protein was completely-sequenced. The translated sequence yields a protein of 566 amino acids with a calculated molecular mass of 61 kDa, suggesting that carbohydrates represent about 50% of the mass of the protein.

Differential proliferative response of human and mouse astrocytes to gamma-interferon.

We have previously shown that gamma-interferon promoted the proliferation of adult human astrocytes isolated from brain biopsy specimens. In contrast, in the present study, astrocytes derived from neonatal mouse brains and treated with recombinant murine gamma-interferon responded by a decrease (average of 50% at 100 U/ml) in proliferation. The basal rate of proliferation as assessed by bromodeoxyuridine incorporation was markedly increased in neonatal mouse astrocytes when compared to the adult human cells, suggesting that age, and the corresponding metabolic activity of cells, could be important determinants in the mitogenic response of astrocytes to cytokines. However, subsequent examinations of fetal human and adult mouse astrocytes, with comparable basal rate of proliferation to neonatal mouse and adult human cells respectively, showed gamma-interferon to promote DNA synthesis in fetal human astrocytes while inhibiting that of adult mouse astrocytes. The results suggest species differences in the proliferative response of human and mouse astrocytes to the cytokine gamma-interferon.

Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes.

Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (greater than 10(4) transformants per micrograms DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.

Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.