Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.     

Nestmate recognition in the leaf-cutting antAtta laevigata

Summary We tested matureAtta laevigata colonies in the field to see if the ants used queen substances, environmental odours (in this case odours produced by the nest's fungi), an odour produced by each individual, or a gestalt odour (resulting from odours distributed between nestmates) as a discrimination signal for nestmate recognition. We found that nestmate recognition inA. laevigata appears to be largely based on an odour produced by each nestmate which appears to be concentrated in the head, although other odours may also be used. We found no evidence of genetic relatedness influencing the discrimination ability, nor did ants respond differently to neighbors in comparison to non-neighbors.     

Free—living fathead minnows rapidly learn to recognize pike as predators

Individuals from a natural population of approximately 20 000 fathead minnows from a pike–free pond did not respond with appropriate anti–predator behaviour upon encountering pike odour in laboratory tests. However, 14 days after 10 pike were stocked into the pond, minnows had acquired recognition of pike odour. Laboratory studies have indicated several possible mechanisms for acquiring predator recognition in fathead minnows. This study indicates that these, or similar processes, can produce major changes in predator recognition in the wild.     

Preparation,characterization, and structure of half gap junctional layers split with urea and EGTA

Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60–90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca²⁺ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.The antibodies used to analyze the protein composition in our samples were the generous gift of Dr. David Paul. We thank Dr. Camillo Peracchia for sending us a preprint of his chapter on molecular mechanisms of connexon gating and docking which helped guide our thinking about interconnexon forces and John Badger for information on divalent cation sites in protein structure. We thank Amani Thomas-Yusuf for technical assistance. The photographic expertise of Marie Craig is gratefully acknowledged. This work was funded by National Institutes of Health GM43217 to G.E.S. and GM18974 to D.A.G.     

On-line state recognition in a yeast fed-batch culture using error vectors

The physiological states with respect to cell growth and ethanol production in a yeast fed-batch culture expressed in linguistic form could be recognized on-line by fuzzy inferencing based on error vectors. The error vector was newly defined here in a macroscopic elemental balance equation. The physiological states for cell growth and ethanol production were characterized by error vectors using many experimental data from fed-batch cultures. Fuzzy membership functions were constructed from the frequency distributions of the error vectors and state recognition was performed by fuzzy inferencing. In particular, an unusual physiological state for a yeast cultivation, in which aerobic ethanol production was accompanied by very low cell growth, could be recognized accurately. According to the results of the state recognition, an energy parameter, the P/O ratio in the metabolic reaction model was adaptively estimated, and the cell growth was successfully evaluated with the estimated P/O. (c) 1995 John Wiley & Sons, Inc.     

Trypsin complexed with α1-proteinase inhibitor has an increased structural flexibility

Mutant rat trypsin Asp¹⁸⁹Ser was prepared and complexed with highly purified human α₁-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with α₁-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg¹¹⁷-Val¹¹⁸ one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.     

Specific induction of self-discrimination by follicle cells in Ciona intestinalis oocytes

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.     

Arginine side chain assignments in uniformly 15N-labeled proteins using the novel 2D HE(NE)HGHH experiment

A novel 2D NMR experiment, 2D HE(NE)HGHH, is presented for the assignment ofarginine side chain ¹H and ¹⁵N resonances inuniformly ¹⁵N-labeled proteins. Correlations between¹H, ¹Hand ¹H are established on the basis of³J(¹⁵N,¹H) heteronuclear scalarcoupling constants, and sequence-specific assignments are obtained by overlapof these fragments with ¹H chemical shiftsobtained by assignment procedures starting from the polypeptide backbone.Since guanidino protons exchange quite rapidly with the bulk water, the 2DHE(NE)HGHH pulse scheme has been optimized to avoid saturation and dephasingof the water magnetization during the course of the experiment. As anillustration, arginine side chain assignments are presented for two uniformly¹⁵N-labeled proteins of 7 and 23 kDa molecular weight.