Alternative identification test relying upon sexual reproductive abilities of Candida lusitaniae strains isolated from hospitalized patients.

The in vitro mating ability of Candida lusitaniae (teleomorph Clavispora lusitaniae) clinical isolates has been investigated. Studying the effects of culture conditions, we showed that ammonium ion depletion in the medium is a major trigger of the sexual cycle. Moreover, a solid support is required for mating, suggesting a role for adhesion factors in addition to the mating type gene recognition function. Monitoring of mating and meiosis efficiency with auxotrophic strains showed great variations in ascospore yields, which appeared to be strain and temperature dependent, with an optimal range of 18 to 28 degrees C. The morphogenetic events taking place from mating to ascospore release were studied by scanning and electron microscopy, and the ultrastructure of the conjugation canal, through which intercellular nuclear exchanges occur, was revealed. Labeling experiments with a lectin-fluorochrome system revealed that the nuclear transfer was predominantly polarized, thus allowing a distinction between the nucleus donor and the nucleus acceptor strains. The direction of the transfer depended on the strain combination used, rather than on the genotypes of the strains, and did not appear to be controlled by the mating type genes. Finally, we demonstrated that all of the 76 clinical isolates used in this study were able to reproduce sexually when mated with an opposite mating type strain, and we identified a 1:1 MATa/MATalpha ratio in the collection. These results support the idea that there is no anamorph state in C. lusitaniae. Accordingly, the mating type test, which is easy to use and can usually be completed within 48 h, is a reliable alternative identification system for C. lusitaniae.

evx1 transcription in bony fin rays segment boundaries leads to a reiterated pattern during zebrafish fin development and regeneration.

The dermoskeleton of zebrafish fins is composed of actinotrichia and segmented bony rays, or lepidotrichia, which grow by successive addition of distal segments. The present study shows that evx1, a new zebrafish even-skipped related gene (Thaëron et al., 2000) displays during bony ray morphogenesis, a unique repetitive expression pattern along the proximodistal axis of the fin. Whole-mount in situ hybridization performed on larvae and adult regenerating fins show that evx1 signal appears as parallel dash lines crossing the width of each developing and regenerating rays, in a ladder-like fashion. Cytological studies show that a subpopulation of bone forming cells (scleroblasts) expresses evx1 at the level of the joint between two adjacent segments except in the apical part of the differentiating ray where evx1 expression precedes the formation of the joint. This distal transcription is turned on again only when the latest differentiating segment reached its final size and might label the putative next segment boundary. This suggests the existence of a molecular mechanism controlling the periodic expression of evx1 which could be involved in the establishment of segment boundaries during fin ray morphogenesis, and could play a key role during dermal skeleton patterning.

Expression of two even-skipped genes eve1 and evx2 during zebrafish fin morphogenesis and their regulation by retinoic acid.

Growth and patterning during fin regeneration depend, like for fin development, on the integrated expression of homeogenes. In the present work we have studied, by in situ hybridization, the expression and regulation of two vertebrate homologs eve1 and evx2 of the Drosophila pair-rule even-skipped gene family. Upon amputation of pectoral and caudal fins, both genes, expressed transiently in the mesenchyme during early stages of fin development of these fins, are turned on. During the formation of the blastema they are transcribed first in the mesenchyme located underneath the wound epidermis and then, their expression is restricted to the regenerating rays regions. These expression patterns are developmentally regulated since both genes are no longer transcribed when the bony rays are differentiating. Exposure of the regenerates to retinoic acid (RA) modifies the boundaries of eve1 and evx2 expression: the signal is down-regulated in the ray region and up-regulated in the interray region. Moreover, expression is induced in the wound epidermis. These results indicate that eve1 and evx2 products are part of the molecular signals involved in pattern formation of the fin and fin rays in connection with outgrowth. RA might alter growth and morphogenesis of the regenerating fins by a fine regulation of these genes among others.

Caudal fin regeneration in wild type and long-fin mutant zebrafish is affected by retinoic acid.

Zebrafish (Danio rerio) represents an ideal experimental model to tackle fundamental issues concerned with organogenesis during development and regeneration of complex body structures. We discuss here the development of the skeleton in zebrafish caudal fins, their regenerative ability in wild type and long-fin mutant adult fish, and how retinoic acid (RA), which induces duplications along the proximodistal axis in regenerating limbs, affects regeneration of the caudal fin. The dorsal and ventral lobes of zebrafish caudal fins are apparently symmetrical along the dorsoventral axis, but all of the skeletal elements and most of the soft tissues of both lobes originate from the ventral part of the embryo, as demonstrated by whole-mount staining of developing fish. Analysis of caudal fin regenerates in wild type adults does not reveal any difference in the regenerative ability of the two lobes, and in the length of the regenerate in comparison with the amputated part. In contrast, in the long-fin mutant the regenerated caudal fin is always somehow defective in that the original asymmetry in the length of the two lobes observed in this mutant is not reproduced in the regenerate. Furthermore, in the majority of the batches studied the regenerate is much smaller in size than the amputated part. This suggests that this mutant may be valuable to further our understanding of the mechanisms underlying growth control and patterning during regeneration. Finally, we show that the regenerating caudal fin is sensitive to RA-treatment, and clear teratogenic effects on the dorso-ventral axis are observed under many of the experimental conditions investigated both in wild type and long-fin mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of c-myc expression during oogenesis and embryonic development in Xenopus laevis.

The expression of the proto-oncogene c-myc during oogenesis and embryonic development was followed by in situ hybridization using a cytological protocol adapted to amphibian embryos. The c-myc RNA was highly expressed in the cytoplasm of young oocytes and was further diluted during oocyte growth without specific localization. From the neurula stage on, new myc transcripts were detected and the whole embryo appeared positive with antisense myc RNA probes relative to control sense RNA probes. In addition, a spatial localization of high levels of the transcript was also observed in specific areas of the developing embryo, including the epidermis, gill buds, optic vesicles and lens placodes. These observations might indicate a specific role of the c-myc gene during the differentiation of these tissues. Alternatively, this high level of myc expression might prevent such tissues from entering into terminal differentiation during the growth of the embryo.

Proto-oncogenes and embryonic development.

The role of proto-oncogenes in embryonic development was investigated using one of the most characterized vertebrates, the amphibian Xenopus laevis. Genes which belong to the major proto-oncogene families have been detected in Xenopus genome. The developmental control of the myc gene was assayed using a characterized Xenopus myc probe and specific antibodies. The myc gene is highly expressed as a stable maternal mRNA in oocyte, and an unfertilized egg contains 5 X 10(5)-fold the myc RNA content of a proliferative somatic cell. The myc RNA store is evenly distributed in the oocyte and the egg. Fertilization triggers a post-transcriptional control of the gene and the RNA store is progressively degraded to a constitutive value of 10 to 30 myc RNA copies registered per gastrula embryonic cell. The 62K myc protein is accumulated late in oogenesis. This uncoupling of myc expression and cell proliferation appears as a specific developmental regulation of the myc gene, adapted to the series of rapid cell cleavages occurring after fertilization.

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system. II. The effect of 60 Hz electric fields on the growth of different regions of the cucurbit root elongation zone.

The region of elongation in Cucumis sativus and Cucurbita maxima roots was marked at increasing distances from the apex to provide an analog of increasing cell size. These roots were exposed/sham-exposed to 60 Hz electric fields and the growth rates of the root segments measured. The growth rate effect magnitude varied with increasing distance from the root tip at constant field strength, and with increasing applied field strength. These results provide strong, qualitative support for the postulate that ELF transmembrane potential induction is involved in the stimulation of ELF electric field effects in the plant root model system.

Diagnostic ultrasound and sister chromatid exchanges: failure to reproduce positive findings.

Human lymphocytes were exposed in vitro to ultrasound from two clinical devices, one of which was previously reported to have increased the frequency of sister chromatid exchanges. The ultrasonic exposures had no significant effect on the frequency of sister chromatid exchanges from three blood donors. Exposure to ultrasound also had no effect on cell cycle progression. A concomitant positive control (mitomycin C) resulted in a significant increase in sister chromatid exchanges.

A cytohistological analysis of roots whose growth is affected by a 60-Hz electric field.

Roots of Pisum sativum were exposed for 48 h to 60-Hz electric fields of 430 V/m in an aqueous inorganic growth medium. The growth in length of the exposed roots was 44% of that for control roots. Root tips were analyzed for mitotic index and cell cycle duration. Mature, differentiated root sections from tissue produced after electrode energization were analyzed for cell lengths and number of files. The major reason for the observation that exposed roots are shorter than control roots is that cell elongation in the former is greatly diminished relative to controls.

Lack of ultrasound effect on in vitro human lymphocyte sister chromatid exchange.

The frequency of sister chromatid exchanges (SCEs) in human lymphocytes cultured in vitro was not affected by a 30 min exposure to a 2.25 MHz focused ultrasound beam (from a clinical diagnostic unit with a pulse repetition rate of 1000 Hz, a 1 mu sec burst duration, and a 2-200 W/cm2 maximum intensity). A 30 sec exposure to continuous wave 1 MHz 2 W/cm2 (SP) ultrasound from an experimental device lysed 10-15% of the lymphocytes; there was no increase in SCEs in the survivors relative to unexposed controls. Treatment of lymphocytes with 0.033 micrograms/ml mitomycin-C, a known SCE inducer, increased the frequency of SCEs about 4 times above control levels.

Diagnostic insonation of extra utero human placentas: no effect of lymphocytic sister chromatid exchange.

Freshly delivered human placentas were exposed to ultrasound for 30 min using a diagnostic linear array unit. Blood was then drawn and cultured in the presence of bromodeoxyuridine, and the frequencies of sister chromatid exchanges (SCE) in the lymphocytes determined. There was no statistically significant difference in SCE frequencies between control and exposed cells; the frequencies of SCEs per cell ranged from 4.50 to 6.02 for control and from 4.66 to 6.10 for exposed cells in five separate experiments. Positive control mitomycin C treated cells were significantly affected, with more than 50 SCEs per cell.

Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems.

The synchronized divisions following a treatment with hydroxyurea (HU) - an inhibitor of DNA synthesis - were studied in root meristems of Allium sativum using two methods: autoradiography of median sections and morphological labeling with a cytokinesis inhibitor. It is shown that the second wave of mitoses is heterogeneous: it is composed mostly of cells which have been synchronized in the S phase by the HU treatment, of cells coming from the quiescent center stimulated to enter DNA synthesis and of cells which were not blocked by the 23 h HU treatment (slow cycling cells). It is also shown that the cell cycle following the first synchronized division is considerably shortened by the synchronization procedure.

[Study on a synchronized population of the antimitotic effect of cycloheximide at various phases of the cell cycle in Allium sativum L]. / Etude, sur population synchronisée, de l'effet antimitotique du cycloheximide à différentes phases du cycle cellulaire chez Allium sativum L.

The effect on the entry in mitosis, induced in synchronized meristematic cells of Allium sativum L. by a treatment with cycloheximide (10 mug/ml-30 min) at different phases of the cell cycle has been investigated. In all cases cells enter mitosis after a long delay but it appears that the delay induced on G1 cells is longer. Incorporation of 3H leucine is immediately inhibited at any stage of the cell cycle.

[Cytotoxicity of aurintricarboxylic acid: inhibition of cell proliferation and of protein synthesis in Allium sativum L. root meristems]. / Sur la cytotoxicité de l'acide aurine-tricarboxylique: inhibition de la prolifération cellulaire et des synthèses protéiques dans les méristèmes radiculaires d'Allium sativum L

The inhibitory action of aurine-tricarboxylic acid (ATA) on protein synthesis, known in vitro, has been checked in vivo in Allium sativum L. root meristems. Parallel to this inhibition, ATA acts on cell proliferation, on the one hand by preventing the entering of cells into prophase and, on the other hand, by disturbing the process of mitosis.