Developmental changes in cyclin B1 and cyclin-dependent kinase 1 (CDK1) levels in the different populations of spermatogenic cells of the post-natal rat testis

Spermatogenesis is a highly ordered process which requires mitotic and meiotic divisions. In this work, we studied the relative changes in the levels of the two components of the M-phase promoting factor (MPF): the regulatory subunit cyclin B1 (CycB1) and its catalytic subunit cdk1, in spermatogenic cells of rats between 16 and 90 days of life. A multivariate flow cytometry analysis of forward scatter (FSC), side scatter (SSC) and DNA content was used to identify six populations of rat germ cells: spermatogonia with preleptotene spermatocytes, young pachytene spermatocytes, middle to late pachytene spermatocytes, secondary spermatocytes with doublets of round spermatids, round spermatids, and elongated spermatids. For any population studied no significant difference in the relative cellular content of CycB1 or cdk1 proteins between animals of different ages was observed. By contrast, CycB1 and cdk1 levels were different between the different populations of germ cells. CycB1 and cdk1 were rather high in young pachytene spermatocytes and culminated in late spermatocytes, i.e. just before the first meiotic division. The relative levels of the two proteins remained high in secondary spermatocytes then decreased in round spermatids at the exit of meiosis. Similar results were obtained by Western-blot analysis of total proteins obtained from lysates of elutriated fractions of spermatocytes and spermatids. MPF activity was assessed in lysates of germ cells from 32-day-old rats or adult animals using p13suc1 agarose and histone H1 as an exogenous substrate. H1 kinase activity was higher in pachytene spermatocytes than in round spermatid fractions from both adult and young rats. These results indicate that the meiotic G2/M transition is associated to high levels of CycB1 and cdk1 leading to high MPF activity irrespective of the age of the animals.     

Microgravity and hypergravity effects on fertilization of the salamander Pleurodeles waltl (urodele amphibian)

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.     

Nitrogen excretion and changes in blood components during emersion of the subtidal spider crab Maia squinado (L.)

Survival ability of Maia squinado to emersion and subsequent reimmersion was determined in winter and summer conditions. Male spider crabs were less tolerant of emersion than females. Emersion (up to 24 h in summer and to 48 h in winter) induced a marked reduction of nitrogen excretion, especially ammonia excretion. Increase in blood ammonia content was rapid and very high in summer (1750 micromol l(-1)), but non-lethal levels. Estimation of the body ammonia overload showed that only 30% of unexcreted ammonia accumulated in blood. The ammonia release at reimmersion indicated that ammonia also accumulated in other body compartments. Increase in blood urate content, which indirectly reduces ammonia production, was similar at both seasons. Emersed M. squinado was rapidly resorting to anaerobic metabolism, especially in summer when its blood haemocyanin content is low. A strong hyperglycemia was developed in the first 12 h of emersion at both seasons. Mortality occurring beyond 24 h of reimmersion, when the body ammonia overload is cancelled and the recovery of most of blood components is achieved, remains unexplained.     

Spectroscopic studies on ethidium bromide binding to intramolecular parallel and antiparallel triple helices containing T*A:T and G*G:C triplets

The interaction of ethidium bromide (EB), a DNA intercalator, with two intramolecular triplexes 5'd(G4A4G4-[T4]-C4T4C4-[T4]-G4T4G4), 5'd(G4T4G4-[T4]-G4A4G4-[T4]-C4T4C4) ([T4] represents a stretch of 4 thymine residues) and their precursor duplexes has been investigated by circular dichroism, fluorescence and UV absorption spectroscopy. Binding of EB induces a circular dichroism band in the region around 310 nm which is positive for the duplex forms but negative for the triplex forms. We observed that the binding of EB to the duplex form does not induce the formation of the triplex structures. Thermal denaturation experiments demonstrate that EB stabilizes more the parallel triple helix than the antiparallel one. Analysis of the binding process from fluorescence measurements shows that binding constants to the triple helical forms and to the hairpin reference duplex [T4]-G4A4G4-[T4]-C4T4C4) are close. However the binding site size is larger for the triplexes (4-6 base triplets) than for the duplex (2 base pairs).     

Distribution and palaeoecology of freshwater algae and stromatolites: III, some new forms from the Carboniferous, Permian and Triassic of France and Spain

New collections allow us to describe some new algae forming: a) centimetric, unorganized, masses (Toutinella bifurcata from the Middle Triassic of Provence); b) large, decimetric masses (Zarramanella menorica from the ‘Buntsandstein’ of Minorca, Balearic Islands) and c) interesting, plurispecific biocenosis, laminated and silicified, from the French Massif Central: Lower Stephanian of Saint-Étienne basin (with Hankerpella minima, Baltzerella stephaniense, Ponsinella undulata) and Lower Autunian of Bourbon-l'Archambault basin (Hanskerpella pustulata, Baltzerella stephaniense, Ponsinella multifrons, Sarfatigirella intricata). Other forms are badly preserved, one is possibly a Broutinella sp. and another may be a coccoid form of cyanobacteria or chlorophyceae. Silicified biocenoses macroscopically occur in the form of regular laminations, exhibiting vertical, planar joints (1–2 cm wide) and horizontal, planar joints, 5 mm to 3 cm thick, with various sedimentary infillings. These facies represent early and late silicifications of biological felts (mats), probably previously partly calcified and affected by desiccation cracks partly infilled before the silicification. There is no evidence for an early silicification of living filaments and trichomes. The silicification also affects other types of sediments (petites, sandstones, conglomerates, breccias) associated with the algal formations. Macroscopically, these laminated formations do not show domes or hemispheres; on the other hand, these features clearly appear under the microscope, and these formations are true stromatolites. The basins of Saint-Étienne and Bourbon-l'Archambault contain volcanic rocks; the siliceous series, very localized in the basins, represent an impregnation of various primary materials by silica of hydrothermal origin. The present study is voluntarily limited to the palaeontological and palaeoecological appearances; algal laminations formed in ephemeral pools and small lakes, under very particular conditions, ‘hydrothermal’ sensu lato, are still to be stated more precisely.

Résumé

Des récoltes nouvelles permettent de décrire quelques algues nouvelles formant des amas centimétriques non organisés (Toutinella bifurcata du Trias moyen de Provence), de grosses masses décimétriques (Zarmmanella menorica du Buntsandstein de Minorque) et d'intéressantes biocrenoses plurispécifiques laminées et silicifiées du Massif central français: Stephanien inférieur de Saint-Étienne (à Hankerpella minima, Baltzerella stephaniense, Ponsinella undulata), Autunien inférieur du bassin de Bourbon-l'Archambault (Hanskerpella pustulata, Baltzerella stephaniense, Ponxinella multifrons, Sarfatigirella intricata). D'autres formes sont mal conservées, l'une serait attribuables à une ? Broutinella sp. et une autre à une coccoide (cyanobactéries ou chlorophycées). Les biocœnoses silicificées se présentent macroscopiquement sous forme de laminations régulières, accidentées de fentes planes verticales (1–2 cm de largeur) et de fentes planes horizontales (5 mm–3 cm de hauteur), à remplissages sédimentaires variés. Ces faciès correspondent à des silicifications précoces et tardives de feutres biologiques probablement en grande partie calcifiés précocement et affectés de fentes de dessiccation partiellement remplies avant la silicification. Aucun indice de silicification de filaments et de trichomes du vivant des organismes n'a pu être observé. La silicification affecte aussi d'autres sédiments (pélites, grès, conglomérats, brèches), associés aux anciennes formations algaires. Macrospiquement, ces formations laminées ne montrent pas de dômes ni d'hémisphères; en revanche ces structures apparaissent parfaitement au microscope, et ce sont de véritables stromatolites. Les bassins de Saint-Étienne el de Bourbon-l'Archambault contiennent des formations volcaniques; les formations siliceuses, qui sont très localisées dans les bassins, correspondent certainement à une imprégnation par de la silice d'origine hydrothermale de sédiments variés. La présente étude se limite volontairement à l'aspect paléontologique et paléoécologique; les laminations algaires se sont formées dans des bassins ou de petits lacs éphémères, dans des conditions très particulières, hydrothermales sensu lato, encore à préciser.     

Genetic and morphological heterogeneity in small rodent whipworms in southwestern Europe: characterization of Trichuris muris and description of Trichuris arvicolae n. sp. (Nematoda: Trichuridae)

Genetic and morphological variability of whipworms Trichuris Roederer, 1761 (Nematoda: Trichuridae), parasites of small rodents in southwestern Europe, was studied. Isozyme patterns of natural populations of nematodes parasitizing rodent species of the Muridae (Apodemus sylvaticus, Apodemus flavicollis, Mus musculus) and Arvicolidae (Clethrionomys glareolus, Microtus agrestis, Microtus arvalis) were analyzed at 6 putative loci. Two diagnostic loci were found in T. muris from Muridae and from Arvicolidae. Thus, the existence of 2 species of Trichuris restricted to different host families was indicated. They included Trichuris muris Schrank, 1788, originally described as being from mice, and Trichuris arvicolae n. sp., parasitizing the above species of Arvicolidae. The morphological variability of both species was compared. Although ranges of all morphological characters of the new species overlapped with those of T. muris, stepwise discriminant analysis yielded a 100% accurate classification of females when using vagina length and egg size. Males of T. muris and T. arvicolae cannot be separated entirely. A set of 6 variables yielded 95.7% discrimination; the most discriminating variables were spicule size and body width.     

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.     

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m² solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m² artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different. This revised version was published online in June 2006 with corrections to the Cover Date.