Transcription of a zebrafish gene of the hairy-Enhancer of split family delineates the midbrain anlage in the neural plate

her5 encodes a basic helix-loop-helix (bHLH) protein with all features characteristic of the Drosophila hairy-E(spl) family. her5 is expressed in a band of cells within the neural anlage from about 90% epiboly on to at least 36 h postfertilization (hpf). After completion of brain morphogenesis, her5-expressing cells are located in the caudal region of the midbrain, at the boundary with the rhombencephalon. Labelling of cells within the her5 expression domain in the neural plate by injection of fluorescein-dextran allows their labelled progeny to be localized in the 36-hpf-old embryo using an anti-fluorescein antibody. This shows that the her5 expression domain corresponds to the midbrain primordium, including both the tectum and the tegmentum, in the neural plate. A possible function for her5 in regionalization of the brain and/or control of the midbrain-hindbrain boundary is discussed.     

Effect of water-miscible aprotic solvents on kyotorphin synthesis catalyzed by immobilized α-chymotrypsin

Summary Immobilized -chymotrypsin was used as catalyst to synthesize a kyotorphin derivative (Bz-Tyr-Arg-OEt) in the presence of five water-miscible aprotic solvents (dimethylsulphoxide, dimethylformamide, acetonitrile, acetone and tetrahydrofurane) at 30 °C. By using a kinetically-controlled approach, the maximum synthetic activity was obtained when Arg-OEt was used as nucleophile donor at a concentration 1.5-times higher than the acyl-acceptor substrate (Bz-Tyr-OEt). The water-miscible aprotic solvents enhanced greatly the synthetic activity proportionally to their hidrophilicity properties adequately measured by the log P parameter. At the optimum solvent concentration for the enzymatic peptide synthesis, both the water activity (Aw) of the media and the water content of the immobilized derivative showed a saturation profile against the log P parameter. As a function of the solvent hydrophilicity, these water parameters were shown as key parameters for the increase in the synthetic activity of the enzyme by the presence of these solvents.     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Growth, proline accumulation and water relations of NaCl-selected and non-selected callus lines of Dactylis glomerata L

Sodium chloride-tolerant calli were selected from leaf-derived embryogenic calli of Dactylis glomerata L. on agar solidified medium supplemented with 200 mM NaCl, a concentration lethal to non-selected calli. Growth characteristics, water relations and proline accumulation pattern were compared in selected and non-selected lines. The objective was to gain an understanding of the mechanism(s) of tolerance in the NaCl-tolerant line. Growth in the selected line, as expressed in terms of tolerance index (ratio of fresh wt. on NaCl medium:fresh wt. on NaCl free medium x 100), was greater than that of the non-selected line at all levels of NaCl between 50 and 300 mM. There was no significant difference in proline accumulation in the selected and non-selected lines. Maintenance of turgor by osmotic adjustment was observed in the non-selected line despite decreased growth. In contrast, the selected line lost either the need or the ability to adjust osmotically. There was little or no increase in symplastic osmolality in the selected line when exposed to NaCl. Presumably, selection was made for a salt-excluding tissue that has lost the ability to accumulate solutes and adjust turgor with NaCl stress.     

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.     

Cell competence forAgrobacterium-mediated DNA transfer inPisum sativum L.

Distribution and properties of pea (Pisum sativum L.) cells, competent forAgrobacterium-mediated transformation were analysed byin situ histochemical detection of GUS (-glucuronidase) activity, 4 d after inoculation with engineeredAgrobacterium tumefaciens. The vector system consisted of the hypervirulent disarmed strain EHA101 and the binary plasmid pIBGUS, carrying an intron-containing, 35S-promotor drivengusA (oruidA) gene and two selectable marker genes. Cells competent for transformation were mainly restricted to the dedifferentiating cells neighbouring the vascular system of cotyledon and epicotyl explants. A standardized assay was developed, allowing determination and quantification of factors influencing number and distribution of competent cells. In etiolated seedlings, competence for transformation decreased with the distance of the epicotyl explant from the shoot apex and was specifically induced by the exogenous application of auxins. Transient expression ofgusA afterAgrobacterium-mediated DNA transfer was dramatically reduced upon application of cell-cycle and DNA replication inhibitors aphidicolin, colchicine and nalidixic acid. GUS expression after direct DNA transfer of double-stranded plasmid DNA (via PEG into protoplasts or via particle bombardment of epicotyl segments) was independent of cell-division/DNA replication.A GUS-positive mutant of EHA101 was constructed to allowin situ analysis of attaching bacteria within the plant tissue. Attachment and invasion was inhibited by well-developed cuticula but was restored after chloroform treatment of the tissue surface. Moreover, no correlation was found between distribution of attaching bacteria and the pattern of transformation-competent cells.     

Batch culture of Candida utilis in a medium deprived of a phosphorous source

Summary Candida utilis var. major NRRL-Y-1084 was grown in a defined medium without a phosphorous (P) source. During the exponential phase, cells divided according to a specific growth rate of 0.32 h^-1, which is lower than the usual rate for a balanced medium (0.4–0.6 h^-1). The relative P content of the biomass decreased from 2.70% to 0.75% over a period of 6 h, including 2 h of cell division arrest. At the end of this period there was another interruption of cell division. After that, multiplication restarted at a considerably lower rate and it deviated slightly from the exponential pattern. The stationary phase began when biomass P content reached 0.4%–0.5%, slowly decreasing afterwards to 0.25–0.20%. Biomass synthesis was less affected than cell division by the relative decrease of endogenous P, the two processes differing partially in their kinetics. Cell lysis started shortly before the stationary phase and affected about 20% of the population by the end of the assay. RNA and P content of the resulting biomass were 2.4% and 0.25% respecitvely, P being mainly incorporated to RNA.The relationship of biomass production to glucose uptake was very low, probably because the marked P deficiency called for an increase in energy consumption for growth and specially for maintenance. Compared with yeasts grown in a balanced medium, 40% increase in glycogen was observed, whereas no mean changes in the content of cell wall carbohydrates (glucan and mannan) and that of true protein were found.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET). Agrentina     

Microscopic anatomy of the ovary ofAlouatta caraya

The microscopic structure of theAlouatta caraya ovary is studied in different ages and reproductive stages. The most significant feature seems to be the presence in adult ovaries of abundant glandular interstitial tissue which occupies both the cortex and medulla. It seems to be derived from the theca interna of atretic follicles. Discrete luteinized masses are present in the medulla in all the ovaries observed. Invaginations of the surface epithelium are seen only in infant and juvenile ovaries. The development of cystic follicles seems to be a common pathway of atresia.     

Allozyme variability and phylogenetic relationships in the cultivated potato (Solanum tuberosum) and related species

Gene frequencies at 13 isozyme loci were determined in three South American taxa of cultivated potatoes [the diploid group (gp.) Stenotomum, the diploid subgroups (subgp.) Goniocalyx, and the tetraploid gp. Andigena ofS. tuberosum], in the diploid weed speciesS. sparsipilum, and in most of the main cultivars now raised in the Northern Hemisphere (the tetraploid gp. Tuberosum ofS. tuberosum). High levels of genetic variability (mean number of alleles per locus, percentage of polymorphic loci, and mean heterozygosity) were detected, being higher in tetraploid potatoes. An equilibrium among the evolutionary factors which increase genetic variability and artificial selection for maximum yield would explain the high uniformity of heterozygosity values we observed in both Andigena (0.36 ± 0.02) and Tuberosum (0.38 ± 0.01) cultivars.—The low value of genetic distance (D = 0.044) between Stenotomum and Goniocalyx does not support the status of species forS. goniocalyx.—In most isozyme loci, the electromorphs of gp. Andigena were a combination of those found in both gp. Stenotomum andS. sparsipilum, suggesting an amphidiploid origin of gp. Andigena from that two diploid taxa. The presence in Andigena of unique electromorphs, which were lacking in both gp. Stenotomum andS. sparsipilum, suggests that other diploid species could be also implied in the origin of tetraploid Andean potatoes. Furthermore, since Andigena were more related to Stenotomum (D = 0.052) than toS. sparsipilum (D = 0.241), the autopolyploidization of Stenotomum individuals and the subsequent hybridization with gp. Andigena may also have occurred. Thus, our study suggests a multiple origin (amphidiploidy, autoploidy, and hybridization at tetraploid level) of gp. Andigena.—Most of the electromorphs of gp. Tuberosum were also found in gp. Andigena; both the direct derivation of that group from the Andean tetraploid potatoes and the repeated introgression provided by breeding programmes could explain this result. However, the allele c of Pgm-B, present in 30 out of 76 Tuberosum cultivars from Northern Hemisphere as well as in 3 Chilean Tuberosum cultivars, lacks in the 258 Andigena genotypes sampled, suggesting that Chilean germplasm could have taken part in the origin of at least the 39% of the potato cultivars from Europe and North America analyzed here.—The distanceWagner procedure provides an estimate of a 30% of heterogeneity in the evolutionary divergence shown by different groups of cultivated potatoes. Diploid groups show a higher (22.5%) evolutionary rate than tetraploids, which can be attributed to both tetrasomic inheritance and facultative autofecundation that exists in Andigena and Tuberosum groups. Thus, artificial selection acting since 10000 years has not resulted in a higher rate of molecular evolution at the isozyme level in the tetraploids.