Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

Seasonal incidence and ecology of the tick Ixodes ricinus (Acari: Ixodidae) on grazing pastures in Western France

A longitudinal survey was carried out during a 2 year period in Western France to assess the infestation level of grazing pastures byIxodes ricinus ticks. Four farms were visited once a month and each of the grazing pastures was sampled in the centre and at the border using the blanket dragging method. A total of 3562I. ricinus (34 adults, 900 nymphs and 2628 larvae) were collected and the infestation was significantly higher during the first year (p<0.0001). The infestation level byI. ricinus varied between grazing pastures and farms. Grazing pastures in the vicinity of forest were more infested than the others, all through the study. The seasonal distribution of ticks showed peaks, with low fluctuations between farms, years and stages. Tick abundance could not be related to vegetation, but only to the vicinity of woods.     

The complete nucleotide sequence of the mitochondrial DNA genome of the rainbow trout,Oncorhynchus mykiss

The complete nucleotide sequence of the mitochondrial DNA of the rainbow trout, Onchorynchus mykiss, has been determined. The total length of the molecule is 16,660 bp. The rainbow trout mitochondrial DNA has the same organization described in eutherian mammals, the clawed frog (Xenopus laevis), and the two fish species, Oriental stream loach (Crossotoma lacustre) and carp (Cyprinus carpio). Alignment and comparison of the deduced amino acid sequences of the 13 proteins encoded by rainbow trout and other vertebrate mitochondrial genomes allowed us to estimate that COI is the most conserved mitochondrial subunit (amino acid identity ranging from 85.6% to 94.8%) whereas ATPase 8 is the most variable one (amino acid identity ranging from 30.8% to 70.4%). Putative secondary structures for the 22 tRNAs found in the molecule are given along with an extensive comparison of tRNA sequences among representative species of each major group of vertebrates. In this sense, an unusual cloverleaf structure for the tRNA^Ser(AGY) is proposed. A stem-loop structure inferred for the origin of the L-strand replication (O_L) and the presence of a large polycytidine tract in the O_L loop is described. The existence of this stretch instead of the usual T-rich sequence reported so far in mammal mtDNAs is explained in terms of a less-strict template dependence of the RNA primase involved in the initiation of L-strand replication. Correspondence to: J.M. Bautista     

Selective separation of tryptic beta-casein peptides through ultrafiltration membranes: Influence of ionic interactions

Peptide separation by selective membrane filtration has numerous potential applications such as production of peptides with biological activities or spectific enrichment in compounds acting as flavoring agents or as growth factors required by the fermentation industry. The retention of peptides arising from tryptic hdroysis of beta-casein using an M5 Carbosep membrane (molecular wieght cutoll = 10,000 D) has been studied. The peptides with known sequences were characterized by their molecular weight, isoelectric point, and hydrophobicity. Our experiments highlighted that their transmission involves mechanisms other than size exclusion as developed elsewhere. The effect of ionic interactions between peptides and membrance has been investgated by vrying pH, ionic strength of bulk, and electric potential of filtering material. The charge of both peptides and membrane plays an important role in the transmission, particularly with small size and high or lkow isoelectric point. Then, peptides with the same sign as the membrane have lower transmission than expected from the size xclusion model, whereas peptides with opposite sign have higher trnsmission than expected, and even higher than 1 with some of them. (c) 1995 John Wiley & Sons, Inc.     

New approach to study fast and slow motions in lipid bilayers: application to dimyristoylphosphatidylcholine-cholesterol interactions.

Natural abundance 13C solid-state nuclear magnetic resonance spectroscopy was used to investigate the effect of the incorporation of cholesterol on the dynamics of dimyristoylphosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. In particular, the use of a combination of the cross-polarization and magic angle spinning techniques allows one to obtain very high resolution spectra from which can be distinguished several resonances attributed to the polar head group, the glycerol backbone, and the acyl chains of the lipid molecule. To examine both the fast and slow motions of the lipid bilayers, 1H spin-lattice relaxation times as well as proton and carbon spin-lattice relaxation times in the rotating frame were measured for each resolved resonance of DMPC. The use of the newly developed ramped-amplitude cross-polarization technique results in a significant increase in the stability of the cross-polarization conditions, especially for molecular groups undergoing rapid motions. The combination of T1 and T1 rho measurements indicates that the presence of cholesterol significantly decreases the rate and/or amplitude of both the high and low frequency motions in the DMPC bilayers. This effect is particularly important for the lipid acyl chains and the glycerol backbone region.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Study of pH and temperature-induced transitions in urate oxidase (Uox-EC1.7.3.3) by microcalorimetry (DSC), size exclusion chromatography (SEC) and enzymatic activity experiments

Purified recombinant urate oxidase (urate oxygen oxidoreductase EC 1.7.3.3. re-Uox) has been studied by means of differential scanning calorimetry (DSC) in correlation with enzymatic activity measurements and size exclusion chromatography. Differential scanning calorimetry curves versus pH show two endothermal effects in the pH range 6-10. The first endotherm reveals a maximum stability between pH 7.25 and pH 9.5 corresponding to a temperature of transition T(m1) of 49.0 degrees C and an enthalpy of transition of 326 kJ mol(-1). This value dramatically decreases below pH 7.25. The behavior of the second endotherm is more complex but the temperature of transition T(m2) is constant between pH 9 and 7.25 and a maximum for the corresponding enthalpy is obtained near pH 8 with DeltaH(2)=272 kJ mol(-1). An optimal pH of 8.0 for the stability of the enzymatic activity at elevated temperature was also found which was in good agreement with calorimetric results. Reversibility of the first endotherm is obtained from 20 to 51.5 degrees C. The calorimetric result is correlated to enzymatic activity, purity by size exclusion chromatography (SEC) and protein concentration measurements. In contrast, for the second endotherm, after heating up to 68.9 degrees C, no reversibility was found. Interaction with structural analogues of urate has been studied by DSC. 8-Azahyooxanthine has only a small effect and caffeine has no effect at all. With 8-azaxanthine, a rapid increase of the T(m1) function of the concentration is obtained. At high concentration T(m1) reached the T(m2) value which remained unaffected.     

A non-linear model of phosphorus flux in the phytoplankton of a temperate eutrophic reservoir

Phosphorus fluxes in phytoplankton, between intracellular andexternal compartments have been measured, in the epilimnion ofatemperate eutrophic reservoir, by means of phosphorusdepletionexperiments performed in situ. Flow rates betweencompartments were plotted against the phosphorus concentrationofthe compartments' origin of the flows and fitted to saturationfunctions.A value of phosphorus cell subsistence quota for the wholecommunity has been computed from the X-intercept values ofeveryfunction. The obtained figures are 10–100 times higher,dependingon the reference value, than those measured running theclassicalsingle species growth response experiments in chemostats.We propose a model using five compartments and a series offlowrates between them in order to simulate the general phosphoruscycling in freshwater phytoplankton. Short term simulation ofphosphorus content in every compartment shows a fluctuatingpatternaround rates of equilibrium, in agreement with the pattern ofvariation observed in situ for the selectedcompartments.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

Complex interaction between different proteinaceous components within the cell-wall structure ofCandida albicans

We have previously described a monoclonal antibody, MAb DC3:H10, which recognized an epitope preferentially expressed on the surface ofCandida albicans germ tubes. In the present study we examined the MAb-reactive material further. Immunoblot analysis of the material purified partially by Sephadex G-200 and DEAE-Sephacel chromatography reacted with antibodies to theC. albicans C3d receptor (CR2). In an ELISA, MAb DC3:H10 captured antigen that was recognized by both anti-CR2 and anti-mp58 fibrinogen binding mannoprotein polyclonal antibodies. The MAb DC3:H10 failed to compete with either of these antisera in an ELISA. Indirect immunofluo-rescence (IIF) analysis showed differences in surface distribution for the MAb DC3:H10, the CR2, and the mp 58 epitopes. Dual labeling IIF experiments showed MAb DC3:H10 binding to be unaffected by binding of fibrinogen or anti-mp58 antibody. However, the binding patterns of MAb DC3:H10 were modified in the presence of anti-CR2 antibody, suggesting a complex interaction between these cell wall components.