Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

The function of the chicken p34CDC2 protein kinase in fission yeast is cold sensitive for cell cycle progression through the G1 phase and temperature sensitive for traversal of mitosis.

The protein kinase p34^cdc2 is required at the onset of DNA replication and for entry into mitosis. The catalytic subunit and its regulatory proteins, notably the cyclins, are conserved from yeast to man. This suggests that the control mechanisms necessary for progression through the cell cycle in fission yeast are conserved throughout evolution. This work describes the characterization of a fission yeast strain that is dependent for cell cycle progression on the activity of the p34^CDC2 protein kinase from chicken. The response of the chicken p34^CDC2 protein kinase to cell cycle components of fission yeast was examined. Cells expressing the chicken p34^CDC2 protein divide at reduced size at 31° C. Cells are temperature sensitive at 35.5° C and die as a result of mitotic catastrophe. This phenotype can be rescued by delaying cell cycle progression at the G1-S transition by adding low concentrations of hydroxyurea. Schizosaccharomyces pombe cells that are dependent on chicken p34^CDC2 are cold sensitive. At 19° C to 25° C cells arrest in the G1 phase, while traversal of the G2-M transition is not blocked at low temperature. Expression of chicken p34^CDC2 in the cold-sensitive G2-M mutant cdc2A21 suppresses the G1 arrest. Received: 14 October 1998 / Accepted: 15 March 1999     

Screening method for cDNAs encoding putative enzymes converting loganin into secologanin by a transgenic yeast culture

A screening method was developed for the detection of enzymes converting loganin to secologanin, a precursor in the biosynthesis of indole alkaloids. The method uses a transgenic yeast culture expressing two cDNAs encoding enzymes involved in the terpenoid indole alkaloid biosynthesis. In the presence of secologanin, the yeast culture produces a yellow compound visible on nitrocellulose. This color change was used to screen a cDNA library of Catharanthus roseus for a putative enzyme converting loganin into secologanin.     

Reconstitution of fibroblast growth factor receptor interactions in the yeast two hybrid system

Fibroblast growth factors (FGF) activate their receptors through the formation of trimolecular complexes, composed of a ligand, a receptor, and a heparan sulfate oligosaccharide, all of which are members of particularly large families capable of multiple interactions in a combinatorial fashion. Understanding this large network of interactions not only presents a great challenge, but is practically beyond the capacity of most classical techniques routinely used to study ligand receptor interactions. We have used the yeast two hybrid system to study protein-protein interaction in the FGF family. Both ligand and receptor ectodomains are properly folded and functional in the yeast. Basic FGF (bFGF) expressed in the yeast dimerizes spontaneously. This self-assembly occurs at low affinity, which can be greatly enhanced by the introduction of heparin, supporting a defined role for heparin in bFGF dimerization. Screening a rat embryo cDNA library with bFGF in the yeast two hybrid system identified a short variant of FGF receptor 1, found most frequently in embryonal and tumor cells and which possesses affinity toward bFGF that is significantly greater than that of the more abundant, full-length receptor. We find the yeast two hybrid system, a most suitable alternative method for the analysis of growth factor-receptor interactions as well as for screening for novel interacting proteins and modulators of FGF and its receptors.     

Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Leuconostoc mesenteroides produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association. Received 06 October 1998/ Accepted in revised form 05 February 1999     

The naturally occurring furanones: formation and function from pheromone to food

Three closely related 4-hydroxy-3(2H)-furanones have been found in a range of highly cooked foodstuffs where they are important flavour compounds with aroma threshold values as low as 20 micrograms kg-1 water (approximately 0.14 mumol l-1). The compounds are formed mainly as a result of the operation of the Maillard reactions between sugars and amino acids during heating but one compound, 5-(or 2)-ethyl-2-(or 5)-methyl-4-hydroxy-3(2H)-furanone, appears in practice to be produced by yeast, probably from a Maillard intermediate, during the fermentation stages in the production of soy sauce and beer. The compounds are also important in the flavour of strawberry, raspberry, pineapple and tomato but the route of biosynthesis is unknown. Two 3-hydroxy-2(5H)-furanones, emoxyfuranone and sotolon, which are produced spontaneously from amino acids such as threonine and 4-hydroxy-L-leucine are major contributors to meaty and spicy/nutty flavours in foods. The biosynthesis of 5-(1,2-dihydroxyethyl)-3,4-dihydroxy-2(5H)-furanone (ascorbic acid, vitamin C) and 5-hydroxymethyl-3,4-dihydroxy-2(5H)-furanone (erythroascorbic acid) from sugars in plants and yeast, respectively, has been characterized to the enzymic level. After treatment with chlorine, humic waters contain a range of chloro-furanones, some of which, particularly 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), are powerful mutagens. The furanones which occur in foods are also mutagenic to bacteria and cause DNA damage in laboratory tests. However, these compounds are, in practice, very effective anti-carcinogenic agents in the diets of animals which are being treated with known cancer-inducing compounds such as benzo[alpha]pyrene or azoxymethane. Two of the food-derived furanones have antioxidant activity comparable to that of ascorbic acid. A biological function has been discovered for some of the furanones besides vitamin C. 5-Methyl-4-hydroxy-3(2H)-furanone is a male pheromone in the cockroach Eurycolis florionda (Walker) and the 2,5-dimethyl derivative deters fungal growth on strawberries and is an important component of the attractive aroma of the fruit. The red seaweed Delisea pulchra (Greville) Montagne produces a range of brominated furanones which prevent colonisation of the plant by bacteria by interfering with the acylated homoserine lactone (AHL) signalling system used by the bacteria for quorum sensing. In addition, these compounds can deter grazing by marine herbivores. It is proposed here that the evolved biological function of a number of furanones is to act as inter-organism signal molecules in several different systems. This has resulted in two coincidental effects which are important for humans. Firstly, the easily oxidized nature of the furanones in general, which is likely to be an important property in their functioning as signal molecules, results in both mutagenic and anti-carcinogenic activity. The balance of these two effects from compounds in the diet has yet to be fully established. Secondly, and more specifically, the 4-hydroxy-3(2H)-furanones associated with fruit aromas act to attract animals to the fruit, which ensures seed dispersal. In the case of humans, the coincidental synthesis of some of these compounds in foods during preparation results in these foods appearing particularly attractive through the transferred operation of the original signalling mechanisms.     

Resistance of Maize Calli to Herbicide Basta and Its Relevant Effect by Some Amino Acids

Maize (Zea mays L. ) embryogenic calli were cultured on N6 medium and treated respectively with different concentrations of herbicide Basra and amino acids. The NH4+ concentrations and weight increase of maize calli were measured. Statistical analysis revealed that callus weight increased less when cultured on N6 medium with 4 mg/L of Basta while Nih+ concentration reached its peak when cultured on N6 with 8 mg/L of Basta. Therefore 6 mg/L of Basta was considered as the optional dosage for selection of transgenic calli L-arginine and L-glutamic acid significantly reduced the NH4+ concentration in maize calli while L-proline had little effects on NH4+ concentration even though it enhanced callus weight enormously.     

Induction of embryogenesis from isolated apple microspores

 We report, for the first time, the induction of embryogenesis and plant formation from isolated apple (Malus domestica Borkh.) microspores in vitro. Different isolation techniques were tested and an optimized protocol was elaborated. Furthermore, the influence of the induction medium and starvation treatment, using different starvation material, temperatures and time, were studied. In addition to embryo induction, the number of multicellular structures per divided microspores was found to be a suitable parameter of assessment and could be used in earlier stages during microspore culture. Although the number of embryos induced in these first experiments is low, the best frequency of embryo induction was shown to be at least twice as efficient as that obtained by anther culture. Received: 9 September 1998 / Revision received: 22 December 1998 / Accepted: 12 January 1999