Cell adhesion glycoprotein vitronectin during Xenopus laevis embryogenesis.

Vitronectin (vn) is a cell-adhesive glycoprotein present in blood and extracellular matrix of all vertebrates. In the present study we reported the cDNA cloning of Xenopus laevisvitronectin and its spatial and temporal expression pattern during the embryonic development of this important model organism. The deduced amino acid sequence of Xenopus laevis vn showed 49%, 47% and 43% identity with human, chicken and zebrafish orthologs, respectively, whereas the comparison with Xenopus tropicalis vn presented 85% identity. The structural organization consisting of a somatomedin B domain and two hemopexin-like domains was similar to higher vertebrate vitronectins. The vn transcripts were detected from stage 28 onward. At tadpole stages, vn is expressed in heart, gut derivatives and in the notochord. The protein was detected in heart, liver, foregut, pronephros and notochord at stages 43 and 47 of Xenopus embryos. Our results suggest that vitronectin is developmentally regulated and could participate in embryo organogenesis.

Ultrastructural changes in the follicular epithelium of Ceratophrys cranwelli previtellogenic oocytes.

In this work we carried out ultrastructural, autoradiographic and biochemical analyses of the follicular epithelium during C. cranwelli previtellogenesis. This study revealed that the follicular epithelium in early previtellogenesis is constituted of a single layer of squamous homogeneous cells. During mid-previtellogenesis two types of cells develop: dark cells and clear cells. The follicular dark cells are actively involved in the synthesis of RNA, which is transferred to the oocyte through the interface. In late previtellogenesis the dark cells show apoptotic characteristics such as chromatin condensation, DNA fragmentation and cytoplasm shrinkage. This process forms apoptotic bodies that seem to be engulfed by the oocyte. Our results show evidence that, during mid- and late C. cranwelli previtellogenesis, the follicular epithelium undergoes remodelling processes interacting with the oocyte.

Implication of gap junction coupling in amphibian vitellogenin uptake.

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

First Report of Colletotrichum gloeosporioides on Strawberry in Northwestern Argentina.

Colletotrichum gloeosporioides was isolated from symptomatic strawberry (Fragaria × ananassa Duch. 'Chandler') growing in Lules (Tucumán, Argentina). Isolates were characterized based on several criteria. Potato dextrose agar (PDA) was used to evaluate cultural and morphological characteristics of the isolates. After 10 days on PDA at 28°C under continuous white light, colonies showed abundant aerial, cottony white to pale beige growth, with orange asexual fruiting bodies in older colonies. Isolates displayed cylindrical conidia, rounded at both ends, averaging 10.4 × 3.9 µm (length by width). A sexual phase (perithecia) was observed in all isolates in 2-month-old cultures on PDA at 28°C under continuous white light. Pathogenicity tests were conducted with healthy plants of cvs. Pájaro and Chandler. Spray inoculation with conidial suspensions (106 conidia per ml) resulted in disease symptoms (petiole and crown lesions with wilting of crown-infected plants) 7 days after inoculation. Infection progressed at a higher rate in Pájaro than in Chandler. Reisolations from infected strawberry lesions yielded isolates with characteristics identical to the isolate used to inoculate the host. Based on morphological and cultural characteristics, isolates were identified as C. gloeosporioides Penz. & Sacc. (teleomorph Glomerella cingulata Spauld & H. Schenk) (1). This is the first report of C. gloeosporioides causing strawberry anthracnose in northwestern Argentina. Reference: (1) P. S. Gunnell and W. D. Gubler. Mycol. 84:157, 1992.

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae.

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25 degrees C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ approximately 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42-51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52-56].

Carbon and energetic uncoupling are associated with block of division at different stages of the cell cycle in several cdc mutants of Saccharomyces cerevisiae.

Cell proliferation arrest at 37 degrees C (restrictive temperature) of the cell division cycle (cdc) mutants of Saccharomyces cerevisiae cdc28, cdc35, cdc19, cdc21, and cdc17 was correlated with carbon and energy uncoupling. At 37 degrees C, cdc mutants diverted to biomass synthesis only 3 to 4% and 8 to 24% of the fluxes of carbon consumed and ATP obtained by catabolism, respectively, compared with 48 and 34% in the wild-type strain A364A. At the permissive temperature (25 degrees C), the wild type showed similar carbon and energy coupling indexes as at 37 degrees C. However, carbon and energy coupling indexes were two- to sevenfold higher at 25 degrees than at 37 degrees C in cdc mutants; e.g., at 25 degrees C two- to sevenfold higher amounts of carbon and ATP were directed to biomass production than at 37 degrees C. The wild-type strain exhibited a purely oxidative glucose catabolism at 37 degrees C (RQ approximately 1.0), while the cell proliferation arrest of cdc mutants at the same temperature was characterized by fermentative metabolism. At 37 degrees C, cdc mutants directed 50 to 60% of the carbon to ethanol production; 3 to 12% of the carbon was recovered as glycerol in cdc mutants as well as in the wild type. The proliferation arrest of the cell division cycle mutant cdc28 correlated with a significant decrease in the incorporation of radioactive precursors into DNA, RNA, and proteins. In the presence of 8-hydroxyquinoline, the wild-type strain underwent cell proliferation arrest and also exhibited metabolic uncoupling with bioenergetic and catabolic behavior similar to that of the cdc mutants at 37 degrees C. Experimental evidence obtained with cdc19, whose defective gene product is pyruvate kinase, suggests that the primary defect of cdc mutants correlates with a metabolically, highly uncoupled yeast cell. The results presented point to the existence of strong carbon and energy uncoupling together with cell division arrest exhibited by cdc mutants at the restrictive temperature. The degree of uncoupling appears to be tuned, at least in part, by the increase in flux of sugar catabolism through the ethanol fermentative pathway.

Carbon and energy uncoupling associated with cell cycle arrest of cdc mutants of Saccharomyces cerevisiae may be linked to glucose-induced catabolite repression.

Several cell division cycle (cdc) mutants of Saccharomyces cerevisiae (cdc28, cdc35, cdc19, cdc21, and cdc17) at the restrictive temperature (37 degrees C) in the presence of 1% glucose and defined medium divert most of the carbon (approximately 50%) to ethanol production with low biomass growth yields (Yglc) that correlate with carbon and energy uncoupling and arrest of cell proliferation. The cdc mutants studied are shown to be glucose-repressed, while this was not the case for the wild-type A364A (WT). At 37 degrees C, in the presence of 1% glycerol, derepressed cdc28 mutant cells did not show arrest of cell division and carbon and energy uncoupling since the Yglc levels measured were similar to those of the WT strain. These results suggest that the increased fermentative ability and carbon and energy uncoupling exhibited in the presence of glucose by cdc mutants with respect to those exhibited by the WT may be due to catabolite repression.