Essential genes for myoblast fusion in Drosophila embryogenesis.

In Drosophila, as in vertebrates, each muscle is a syncytium and arises from mesodermal cells by successive fusion. This requires cell-cell recognition, alignment, formation of prefusion complexes, followed by electron-dense plaques and membrane breakdown. Because muscle development in Drosophila is rapid and well-documented, it has been possible to identify several genes essential for fusion. Molecular analysis of two of these genes revealed the importance of cytoplasmic components. One of these, Myoblast city, is expressed in several tissues and is homologous to the mammalian protein DOCK180. Myoblast city is presumably involved in cell recognition and cell adhesion. Blown fuse, the second cytoplasmic component, is selectively expressed in the mesoderm and essential in order to proceed from the prefusion complex to electron-dense plaques at opposed membranes between adjacent myoblasts. The rolling stone gene is transiently expressed during myoblast fusion. The Rost protein is located in the membrane and thus might be a key component for cell recognition. The molecular characterization of further genes relevant for fusion such as singles bar and sticks and stones will help to elucidate the mechanism of myoblast fusion in Drosophila.     

Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

Phytogeography of the bryophyte floras of oak forests and páramo of the Cordillera de Talamanca, Costa Rica

Aim Central America is a biogeographically interesting area because of its location between the rich and very different biota of North and South America. We aim to assess phytogeographical patterns in the bryophyte floras of oak forests and páramo of the Cordillera de Talamanca, Costa Rica. Location Tropical America, in particular the montane area of Cordillera de Talamanca, Costa Rica. Methods The analysis is based on a new critical inventory of the montane bryophyte flora of Cordillera de Talamanca. All species were assigned to phytogeographical elements on the basis of their currently known distribution. Absolute and percentage similarities were employed to evaluate floristic affinities. Results A total of 401 species [191 hepatics (liverworts), one hornwort, 209 mosses] are recorded; of these, 251 species (128 hepatics, one hornwort, 122 mosses) occur in oak forests. Ninety‐three per cent of all oak forest species are tropical in distribution, the remaining 7% are temperate (4%) and cosmopolitan (3%) species. The neotropical element includes almost 74% of the species, the wide tropical element (pantropical, amphi‐atlantic, amphi‐pacific) only 19%. A significant part of the neotropical species from oak forests are species with tropical Andean‐centred ranges (27%). As compared with bryophyte species, vascular plant genera in the study region are represented by fewer neotropical, more temperate and more amphi‐pacific taxa. Bryophyte floras of different microhabitats within the oak forest and epiphytic bryophyte floras on Quercus copeyensis in primary, early secondary and late secondary oak forest show a similar phytogeographical make‐up to the total oak forest bryophyte flora. Comparison of oak forest and páramo reveals a greater affinity of the páramo bryophyte flora to temperate regions and the great importance of the páramo element in páramo. Surprisingly, oak forests have more Central American endemics than páramo. Main conclusions (1) Providing first insights into the phytogeographical patterns of the bryophyte flora of oak forests and páramo, we are able to confirm general phytogeographical trends recorded from vascular plant genera of the study area although the latter were more rich in temperate taxa. (2) Andean‐centred species are a conspicuous element in the bryophyte flora of Cordillera de Talamanca, reflecting the close historical connection between the montane bryophyte floras of Costa Rica and South America. (3) High percentages of Central American endemics in the bryophyte flora of the oak forests suggest the importance of climatic changes associated with Pleistocene glaciations for allopatric speciation.     

A light and electron microscopic study of the quadriflagellate green algaSpermatozopsis exsultans

The green flagellateSpermatozopsis exsultans Korshikov has been studied in culture by light and electron microscopy. The organism is naked, bears four flagella and is conspicuously spirally twisted. The ultrastructure and location of cell organelles (except the flagellar apparatus) has been investigated in detail using an absolute configuration analysis. With the exception of a doubling of the flagella and of the secondary cytoskeletal microtubule system,S. exsultans has the exact same complement of organelles occupying the same relative positions as has been described forS. similis. The two species are therefore correctly placed in the same genus. The usefulness of absolute orientations of cell organelles for green algal taxonomy and phylogeny is stressed.Dedicated to Prof.M. Mix on the occasion of her 60th birthday.     

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

1. Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. 2. In this study trypsin (30-50 micrograms/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. 3. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsin-sensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. 4. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3-10 micrograms/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.