Systematics of the species of the yeast genus Saccharomyces associated with the fermentation industry

Summary The so-called wine yeasts Saccharomyces cerevisiae, S. chevalieri, S. bayanus, S. italicus and S. uvarum are characterized by high ethanol tolerance and fermentation velocity. They are ecologically related, being predominantly associated with grape must and wine, and are taxonomically indistinguishable. The only significant physiological differences are between the ability to ferment certain sugars. A taxonomic revision of more than 1,000 strains isolated during the past 50 years and belonging to the above species showed extreme instability in the ability to ferment different sugars. The relationships between these yeasts were examined for DNA base composition and DNA-DNA reassociation. The G+C ranged from 37.6% to 39.0% while optical reassociation experiments defined a first group of species (Saccharomyces cerevisiae, S. chevalieri and S. italicus) exhibiting high base sequence complementarity (>90%). S. bayanus and S. uvarum also showed a high degree of relatedness. Low homology values (30%) indicate that the two groups of species are not closely related. While it is proposed to combine S. cerevisiae, S. chevalieri and S. italicus into one single species under the oldest epithet Saccharomyces cerevisiae, a study of a larger number of strains is recommended before considering the taxonomic position of S. bayanus and S. uvarum.     

Taxanes from Taxus x media

The roots of Taxus x media gave two new taxoids, the structures of which were established as 10-deacetyl-10-dehydro-7-acetyl taxol A and 10-deacetylyunnanxane on the basis of spectroscopic data.     

Growth regulation of human glioblastoma t98g cells by insulin-like growth factor-1 and its receptor

The interaction of insulin-like growth factors (IGFs) with the IGF-1 receptor is an important step in the control of cell proliferation and development. In particular, IGF-1 and IGF-2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum-free medium at 34°C, despite their secretion of substantial amounts of bioactive IGF-1. To be stimulated to divide, growth-arrested cells required the addition of platelet-derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum-free medium could also be obtained by shifting the cells to a temperature of 39.6°C. Treatment of growth-arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF-1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF-1 receptor allowed autonomous growth in serum-free medium at 34°C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF-1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF-1 receptor mRNA. These results demonstrate that the IGF-1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF-1 with its receptor regulates both autonomous and anchorage-independent growth of these cells. © 1994 wiley-Liss, Inc.     

An Exploration of the Effects of Constraints on the Phosphorylation of Synthetic Protein Tyrosine Kinase Peptide Substrates

We synthesized by classical solution methods three conformational constrained analogues of EDNEYTA, a heptapeptide sequence that represents the common major autophosphorylation site of the protein tyrosine kinases (PTKs) of the Src family. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as PTK substrates was examined. The kinetic data obtained indicate that the introduction of the tyrosine-analogue constraints Tic(OH) and MeTyr, which block the ring flexibility, completely prevents the phosphorylation catalysed by the kinases Lyn and Fgr. On the other hand PTKIIB/p38^syk can phosphorylate the two derivatives albeit with an efficiency lower than that found with the native sequence. A third derivative contained side chain to side chain cyclization. This analogue, in which the freedom of the phenolic moiety is not altered, can be phosphorylated by all the PTKs tested with kinetic constants comparable to the parent peptide.     

Identification and Phytotoxicity of 3-methylthiopropanoic and Trans-3-methylthiopropenoic Acids Produced in Culture by Xanthomonas campestris pv. vitians

Two phytotoxic metabolites were isolated from culture filtrates of Xanthomonas campestris pv, vitians , the causal agent of lettuce leaf spots and headrot. The two compounds were identified as 3-methylthiopropanoic (1) and trans-3-methylthiopropenoic (2) acids by chemical and spectroscopic methods. Toxic effects of the two compounds on leaf tissues and protoplasts of lettuce and cabbage were investigated. Solutions of 1 and 2 induced chlorosis and necrosis on lettuce leaves at minimum concentrations of 300 and 50 μg/ml, respectively. Infiltration in cabbage leaves did not produce any symptoms. The LD₅₀ values for 1 and 2 against lettuce protoplasts were 15 and 16 μg/ml, respectively. Activity of the two metabolites against cabbage protoplasts was very low (LD₅₀ > 500 μg/ml).     

Apoptosis Dependent Decrease of the Intramembrane Ion Traffic in Cultured Mouse Fibroblasts Shown by Conductivity Dispersion

We have investigated the intramembranal ion traffic in apoptotic 3T6 cells in culture. Apoptosis was induced by various treatments, such as serum deprivation, high density growth and hydrogen peroxide at subnecrotic doses. Cell death was assessed by nucleosomal DNA fragmentation, single cell electrophoresis, immunofluorescence and histological staining. To study the modifications of membrane structure and function, we adopted a well established biophysical strategy based on the measurement of the electrical conductivity of cell suspensions, as a function of the frequency of the electrical field applied to the sample. A comparison between the conductivity of normal and apoptotic cell suspensions shows that programmed cell death causes a decrease of membrane conductivity which indicates a diminished intramembranal ion traffic. Our results strongly suggest that one of the early events in the triggering of apoptosis is represented by an overall reduction of plasma membrane function. Finally, our results are in agreement with the idea that the nucleus is not the sole target of the apoptotic process.     

Multiple Pathways for Apoptotic Nuclear Fragmentation

We analyzed the ultrastructure of apoptotic nuclear fragmentation in U937 cells treated with many different apoptogenic agents. We found that this characteristic apoptotic feature can be achieved through multiple alternative pathways, depending on the apoptogenic inducer, leading to slightly different final nuclear morphologies. In most instances, the irregularly shaped nucleus of U937 rounds up; then, chromatin condenses at the nuclear periphery. Condensed chromatin can form protruding patches, which eventually bud from the nucleus in sealed vesicles through a process which is actin-dependent, since it could be blocked by cytochalasins. Alternatively, chromatin condenses in tiny, nonprotruding crescents, and a cleavage in the nuclear sap forms, beginning from the inner nuclear membrane and growing inward, thus splitting the nucleus. In U937 induced to apoptosis by hydrogen peroxide in the presence of ADP-ribosylation inhibitors, the nuclei fragment in many vesicles before chromatin even begins to condense: chromatin condensation probably occurs as a consequence. While all the apoptotic morphologies described above evolve from interphase cells, a peculiar apoptotic morphology, possibly deriving from mitotic cells, is detected upon oxidative stress, recalling the formation of micronuclei by clastogenic treatments; it shows partially membrane-bound chromatin patches, which look midway between condensed chromosomes and apoptotic condensed chromatin. The existence of these multiple pathways for nuclear fragmentation may indicate an evolutionary convergence, suggesting that this event may play an important physiological role in apoptosis.     

Mechanism of activation of the vav protooncogene

vav is a human locus that appears to be specifically expressed in cells of hematopoietic origin regardless of their differentiation lineage. This gene was first identified as a result of its malignant activation during the course of gene transfer assays (Katzav, S., Martin-Zanca, D., and Barbacid, M. EMBO J., 8: 2283-2290, 1989). In this study, we report the isolation of complementary DNA clones containing the entire coding sequence of the mouse vav protooncogene. Antisera raised against a peptide corresponding to a predicted hydrophilic domain have allowed us to identify the product of the vav gene as a 95,000 Da protein. Analysis of the deduced amino acid sequence of p95vav revealed an amino-terminal leucine-rich region not present in the activated vav oncogene. This region consists of an amphipathic helix-loop-helix followed by a leucine zipper, a structure reminiscent of the carboxy-terminal region of myc proteins and the steroid binding domain of nuclear receptors. In vitro mutagenicity studies have indicated that removal of the amphipathic helix-loop-helix is sufficient to activate the transforming potential of human and mouse vav protooncogenes. vav proteins also possess a cysteine-rich domain whose sequence predicts the formation of two putative metal binding-like domains, Cys-X2-Cys-X13-Cys-X2-Cys and His-X2-Cys-X6-Cys-X2-His. Replacement of some of these cysteine and histidine residues completely abolished the transforming activity of vav genes. Further examination of the alignment of cysteine residues in this region revealed an alternative structure, Cys-X2-Cys-X13-Cys-X2-Cys-X7-Cys-X6-Cys, which is reminiscent of the phorbol ester binding domain of protein kinase C. A similar domain has been recently identified in a second enzyme, diacylglycerol kinase. These structural similarities, along with its expression pattern, suggest that the vav protooncogene codes for a new type of signal-transducing molecule that may play an important role in controlling hematopoiesis.     

Modification of lysine residues ofStaphylococcus aureus α-toxin: Effects on its channel-forming properties

Summary Staphylococcus aureaus -toxin opens an ion channel in planar phospholipid bilayers which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel.     

A predictive study on the beginning of the pollen season for Gramineae andolea europaea L.

Summary On the basis of the results of seven years (1982–1988) of pollen and meteorological monitoring in the atmosphere of Perugia and Ascoli Piceno (central Italy) beginning of pollen season forecasts for Gramineae and Olea europaea L. are reported. The beginning of the pollen season for grass varied between May 2 nd and May 27th while for Olea it varied between May 26 th and June 23rd. By a statistical analysis of these data several significant correlations were found between the onset of the principal period of pollination and the air temperature in the preceding months and the number of ?heat units? required to flower. Utilizing multiple regressions a predictive method of the beginning of pollen season for both the taxa is reported.