In synchronized pigs, the duration of ovulation is not affected by insemination and is not a determinant for early embryonic diversity

The duration of ovulation (i.e., the time interval between the ovulation of the first and the last follicle in a sow during an estrus) is said to be related to embryonic diversity, which in turn is related to embryonic mortality. The relationship between the duration of ovulation and within-litter early embryonic diversity and the influence of insemination on the duration of ovulation were studied. To determine whether ovulation assessment (transrectal ultrasonography) influenced early embryonic development, control sows were not scanned. Multiparous cyclic sows with an exogenously induced estrus were used. Ovulation detection by means of transrectal ultrasonography did not influence fertilization rate, accessory sperm count, early embryonic development or early embryonic diversity, and, therefore, ultrasonography appears to be a worthwhile method for studying the time and duration of ovulation. Insemination did not influence (P>0.05) the duration of ovulation, in sows which ovulated between 39 and 49 hours after hCG injection. The duration of ovulation (mean+/-SD(range)) was 2.4+/-0.7 (1.1 to 4.0) hours in 15 sows which were artificially inseminated at 22 and 30 hours after hCG injection. In 8 noninseminated sows, the duration of ovulation was 2.3+/-0.5 (1.5 to 3.3) hours. The duration of ovulation was not related to embryonic diversity (SD of the number of nuclei or the number of cell cycles of embryos in a litter) at 114 to 121 hours after ovulation. Thus, a difference of up to 3 hours in the duration of ovulation does not seem to be an important determinant of variation in embryonic diversity between sows.     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

Evidence for a Role of Protein Kinase C Substrate B-50 (GAP-43) in Ca2+-Induced Neuropeptide Cholecystokinin-8 Release from Permeated Synaptosomes

Abstract: To study the involvement of the protein kinase C (PKC) substrate B-50 [also known as growth-associated protein-43 (GAP-43), neuromodulin, and F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca²⁺ in a concentration-dependent manner (EC₅₀ of ～10^-5M). Ca²⁺-induced CCK-8 release was maximal at 10⁴M Ca²⁺, amounting to ～10% of the initial 6,000 ± 550 fmol of CCK-8 content/mg of synaptosomal protein. Only 30% of the Ca^a+-induced CCK-8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti-B-50 antibodies were introduced into permeated synaptosomes to study their effect on Ca²⁺-induced CCK-8 release. The N-terminally directed antibodies (NM2), which inhibited PKC-mediated B-50 phosphorylation, inhibited Ca²⁺-induced CCK-8 release in a dose-dependent manner, whereas the C-terminally directed antibodies (NM6) affected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibitors PKC_19–36 and 1 ?(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), which inhibited B-50 phosphorylation in permeated synaptosomes, had no effect on Ca²⁺-induced CCK-8 release. Our data strongly indicate that B-50 is involved in the mechanism of presynaptic CCK-8 release, at a step downstream of the Ca²⁺ trigger. As CCK-8 is stored in large densecored vesicles, we conclude that B-50 is an essential factor in the exocytosis from this type of neuropeptide-containing vesicle. The differential effects of the monoclonal antibodies indicate that this B-50 property is localized in the N-terminal region of the B-50 molecule, which contains the PKC phosphorylation site and calmodulin-binding domain.     

Monitoring of algae in Dutch rivers: does it meet its goals?

Observations on phytoplankton in the lower reaches of the rivers Rhine and Meuse were carried out in the framework of the national monitoring programme of the main water systems in The Netherlands. Seasonal changes in density and species composition were analysed to detect the major variables and to discuss whether this monitoring meets its goals. Phytoplankton reached peak densities of 140 and 65 μg L^-1 Chl a in the Rhine and Meuse, respectively. Overall density was correlated with seasonal variation in water discharge rather than with nutrient concentrations, that are high in both rivers. The position of sampling sites in relation to the downstream development of the plankton was very important, especially in the regulated Meuse. Despite hydrographic and chemical differences between the two rivers, many species, predominantly diatoms and green algae, were shared. The occurrence of the diatom Skeletonema subsalsum in the Rhine and the green algae Neodesmus danubialis, Micractinium pusillum and Pseudotetrastrum punctatum in the Meuse (1992, but not 1996) was interpreted as a feature related, respectively, to the high salinity of the Rhine and specific riverine conditions of the Meuse. In general the potamoplankton was characterised as an opportunistic assemblage exploiting the high nutrient contents and disturbed hydrography of both rivers. Nevertheless, the phytoplankton contributed to the characterisation of the present river communities, ones that differ rather strongly in historic records. Although year to year variation in phytoplankton density is mainly related to variation in water discharge, phytoplankton biomass did increase as a result of eutrophication over a period of decades. The widespread nature of many of today's potamoplankton species in the two rivers render these organisms less useful for indicating short-term changes in water quality. However the importance of phytoplankton in trophic relationships merits its inclusion in the monitoring of riverine ecosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and characterization of 1-SST, the key enzyme initiating fructan biosynthesis in young chicory roots (Cichorium intybus)

A genuine 1-SST (sucrose:sucrose 1-fructosy] transferase, EC 2.4.1.99) was purified and characterized from young chicory roots ( Cichorium intybus L. var. foliosum cv. Flash) by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, anion and cation exchange chromatography. This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)⁻¹. The mass of the enzyme was 69 kDa as estimated by gel filtration. On SDS-PAGE apparent molecular masses of 49 kDa (α-subunit) and 24 kDa (β-subunit) were found. Further specification was obtained by MALDI-TOF MS detecting molecular ions at m/z 40109 and 19 896. These two fragments were also found on a western blot using an SDS-boiled chicory root extract and chicken-raised polyclonal antibodies against the purified 1-SST, indicating that the enzyme is a heterodimer in vivo. The N-terminus of chicory root 1-SST α-subunit was shown to be highly homologous with the cDNA-derived amino acid sequences from barley 6-SFT and a number of β-fructosyl hydrolases (in-vertases and fructan hydrolases). However, chicory root 1-SST properties could be clearly differentiated from those of chicory root 1-FFT (EC 2.4.1.100), chicory root acid invertase (EC 3.2.1.26) and yeast invertase. The enzyme mainly produced 1-kes-tose and glucose from physiologically relevant sucrose concentrations, indicating that this 1-SST is the key enzyme initiating fructan biosynthesis in vivo. However, like chicory root 1-FFT and barley 6-SFT, the enzyme also showed some β-fructofuranosi-dase activity (fructosyl transfer to water) at very low sucrose concentrations. Although sucrose clearly is the best substrate for the enzyme, some transferase and β-fructofuranosidase activity were also detected using 1-kestose as the sole substrate.     

In vitro synthesis of fractofuranosyl-only oligosaccharides from inulin and fructose by purified chicory root fructan:fructan fructosyl transferase

Besides the non-reducing inulin series of gluco-frueto oligosaccharides which are common in Asteraceae, two series of reducing fructofuranosyl-only oligosaccharides occur in overwintering, forced, and sprouting chicory roots. Such inulo-n-oses could be produced by the action of an endo-inulinase on gluco-fructo oligosaccharides. We show here that similar series of inulo-n-oses as found in vivo can be produced by incubating fructose and inulin with a purified fructamfructan tructosyl transferase (FFT; EC 2.4.1.100) from chicory roots ( Cichorium intybus L.). Therefore fructose (but not glucose) can act as an acceptor for FFT. Since the inulo-n-oses only appear in vivo once the endogenous fructose concentration increases, we conclude that they are not the result of a putative endo-inulinase but of FFT activity.     

De-novo synthesis of fructans from sucrose in vitro by a combination of two purified enzymes (sucrose: sucrose 1-fructosyl transferase and fructan: fructan 1-fructosyl transferase) from chicory roots (Cichorium intybus L.)

Although fructans occur widely in several plant families and they have been a subject of investigation for decennia, the mechanism of their biosynthesis is not completely elucidated. We succeeded in purifying a fructan: fructan 1-fructosyl transferase (1-FFT; EC 2.4.1.100) from chicory roots (Cichorium intybus L. var. foliosum cv. Flash). In combination with the purified chicory root sucrose: sucrose 1-fructosyl transferase (1-SST; EC 2.4.1.99), this enzyme synthesized a range of naturally occurring chicory fructans (inulins) from sucrose as the sole substrate. Starting from physiologically relevant sucrose concentrations, inulins up to a degree of polymerization (DP) of about 20 were synthesized in vitro after 96 h at 0°C. Neither 1-SST, nor 1-FFT alone could mediate the observed fructan synthesis. Fructan synthesis in vitro was compared starting from 50, 100 and 200 mM sucrose, respectively. The initiation of (DP > 3)-fructan synthesis was found to be correlated with a certain ratio of 1 kestose to sucrose. The data presented now provide strong evidence to validate the 1-SST/1-FFT model for in-vivo fructan synthesis, at least in the Asteraceae.Abbreviations DP degree of polymerization - 1-FFT fructan: fructan 1-fructosyl transferase - 1-SST sucrose: sucrose 1-fructosyl transferase The authors thank E. Nackaerts for valuable technical assistance. W. Van den Ende is grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants.     

Ultrastructure of Draparnaldia glomerata (Chaetophorales, Chlorophyceae) II. Mitosis and cytokinesis

The mitosis and cytokinesis of Draparnaldia glomerata as examined here by transmission electron microscopy are in many aspects similar to those described earlier for other chaetophoralean algae. The standard chaetophoralean model of the mechanism of mitosis/cytokinesis is described in detail. Characteristic in this pattern is the movement of sets of centrioles towards the nuclear poles followed by a proliferation of extranuclear microtubules at prophase, the (partial) fusion of centrioles with the spindle poles at metaphase and anaphase, the simultaneous separation of chromosomes apparently caused by both spindle elongation and shortening of the chromosomal microtubules at anaphase, the expulsion of the centrioles by daughter nuclei and finally the non–persistent spindle at telophase. Cytokinesis takes place by formation of a cell plate associated with phycoplast microtubules. The possible function of the phycoplast in cytokinesis in Draparnaldia is discussed.     

Fermentation of Inulin by Clostridium thermosuccinogenes sp. nov., a Thermophilic Anaerobic Bacterium Isolated from Various Habitats

Four closely related strains of thermophilic bacteria were isolated via enrichment in batch and continuous culture with inulin as the sole source of carbon and energy by using inoculations from various sources. These new strains were isolated from beet pulp from a sugar refinery, soil around a Jerusalem artichoke, fresh cow manure, and mud from a tropical pond in a botanical garden. The cells of this novel species of strictly anaerobic, gram-positive bacteria were rod shaped and nonmotile. Growth on inulin was possible between 40 and 65°C, with optimum growth at 58°C. All strains were capable of fermenting a large number of sugars. Formate, acetate, ethanol, lactate, H₂, and succinate were the main organic fermentation products after growth on fructose, glucose, or inulin. Synthesis of inulinase in batch culture closely paralleled growth, and the enzyme was almost completely cell bound. Strain IC is described as the type strain of a new species, Clostridium thermosuccinogenes sp. nov., with a G+C content of 35.9 mol%.