Karyosystematics of thePhleum alpinum polyploid complex (Poaceae)

The karyotypes of three taxa from thePhleum alpinum group of sect.Phleum (P. alpinum subsp.rhaeticum, 2n = 14,P. commutatum, 2n = 28, and informally namedP. commutatum, 2n = 14) were investigated by Giemsa C-banding. The overall similarity of diploid genomes suggests thatP. alpinum subsp.rhaeticum andP. commutatum are closely related — their karyotypes vary only with respect to their average amounts of telomeric heterochromatin. TheP. commutatum genome contains less telomeric heterochromatin than the genome ofP. alpinum subsp.rhaeticum, but in theP. alpinum group as a whole almost fluent transition between low (1.5%) and high (25.5%) amounts of telomeric heterochromatin was observed among populations. In the karyotype of tetraploidP. commutatum, seven distinguishable chromosome types were observed. Each of them is represented at somatic metaphase by four chromosomes. C-band structure of karyotype and average amount of telomeric heterochromatin suggest that this taxon has originated from hybridization between two related diploid forms of theP. commutatum — P. alpinum complex.     

Entwicklungsgeschichte und Ultrastruktur von Pollenkitt und Exine bei nahe verwandten entomophilen und anemophilen Angiospermensippen derAlismataceae,Liliaceae, Juncaceae,Cyperaceae, Poaceae undAraceae

Some closely related members of the monocotyledonous familiesAlismataceae, Liliaceae, Juncaceae, Cyperaceae, Poaceae andAraceae with variable modes of pollination (insect- and wind-pollination) were studied in relation to the ultrastructure of pollenkitt and exine (amount, consistency and distribution of pollenkitt on the surface of pollen grains). The character syndromes of pollen cementing in entomophilous, anemophilous and intermediate (ambophilous or amphiphilous) monocotyledons are the same in principal as in dicotyledons. Comparing present with former results one can summarize: 1) The pollenkitt is always produced in the same manner by the anther tapetum in all angiosperm sub-classes. 2) The variable stickiness of entomophilous and anemophilous pollen always depends on the particular distribution and consistency of the pollenkitt, but not its amount on the pollen surface. 3) The mostly dry and powdery pollen of anemophilous plants always contains a variable amount of inactive pollenkitt in its exine cavities. 4) A step-by step change of the pollen cementing syndrome can be observed from entomophily towards anemophily. 5) From the omnipresence of pollenkitt in all wind-pollinated angiosperms studied one can conclude that the ancestors of anemophilous angiosperms probably have been zoophilous (i.e. entomophilous) throughout.

     

Optimization of cryopreservation of Artemisia annua L. callus

Cryopreservation of callus tissue of Artimisia annua L. was optimized. Two lines of calli were precultured on MS medium with 5% (v/v) dimethyl sulfoxide, and protected by a cryoprotectant containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, 30% (v/v) glycerol and 13.6% (w/v) sucrose. The highest survival rate of callus A201 reached 87% after it was pretreated at 25°C, cryopreserved by liquid nitrogen, recovered in water bath at 25°C and reloaded at 25°C with 34% (w/v) sucrose solution, and that of callus A202 reached 78% after it was treated as callus A201, except pretreated at 35°C, recovered at 35°C and reloaded with 47.8% (w/v) sucrose solution.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

Ibf-1 (Iodine binding factor), a highly variable marker system in the Triticeae

Summary Isoelectric focusing of extracts from the endosperm of mature grains of hexaploid wheat and related species was used to study the genetic control of Iodine binding factor (IBF). Ten IBF bands were present in Chinese Spring (CS) and analysis of the nullisomictetrasomic and ditelosomic lines of CS showed nine of them to be controlled by genes on the long arms of the homoeologous group 5 chromosomes. Five alleles were detected at Ibf-A1 locus, four at Ibf-B1 and four at Ibf-D1 among a sample of 46 wheat genotypes. Homoeoloci were found on chromosome 5R of Secale cereale, 5E of Agropyron elongatum, 5U of Aegilops umbellulata, 5Agⁱ of Agropyron intermedium, 5S¹ and 4S¹ of Aegilops sharonensis and 4H of Hordeum vulgare.     

Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C

Ten strains representing a novel genus of marine thermophilic archaebacteria growing at between 70 and 103°C with an optimal growth temperature of 100°C and a doubling time of only 37 min were isolated from geothermally heated marine sediments at the beach of Porto di Levante, Vulcano, Italy. The organisms are spherical-shaped, 0.8 to 2.5 m in width and exhibit monopolar polytrichous flagellation. They are strictly anaerobic heterotrophs, growing on starch, maltose, peptone and complex organic substrates. Only CO₂ and H₂ could be detected as metabolic products, the latter being inhibitory to growth at high concentrations. Hydrogen inhibition can be prevented by the addition of S^o, whereupon H₂S is formed in addition, most likely as the result of a detoxification reaction. The GC-content of the DNA of isolate Vc 1 is 38 mol%. The new genus is named Pyrococcus, the fireball. Type species and strain is Pyrococcus furiosus Vc 1 (DSM 3638).     

Random amplified polymorphic DNA variation in the eggplant,Solanum melongena L. (Solanaceae)

RAPD analysis was carried out on 52 accessions of Solanum melongena (eggplant) and related weedy forms known as insanum. Twenty-two primers amplified 130 fragments. Solanum melongena exhibited 117 of the fragments, all of which were also present in insanum. Insanum displayed an additional 13 fragments not found in S. melongena. Overall, the insanum accessions were more diverse than those of S. melongena. The calculated similarity between them was 0.947. The RAPD results were closely concordant with the results of an electrophoretic isozyme survey performed on the same accessions. The concordance of the results shows that even though S. melongena and insanum are highly diverse morphologically, it is no longer appropriate to distinguish them taxonomically.     

Identification of hybrids betweenQuercus petraea andQ. robur (Fagaceae): Results obtained with RAPD markers confirm allozyme studies based on theGot-2 locus

RAPDs were employed as genetic markers to detect interspecific hybridization between the closely related oak speciesQuercus robur andQ. petraea. Fourteen primers were used in order to check the genetic status (pure or hybrid) of individuals classified morphologically. Among the 147 PCR fragments obtained 11 appear to be species-specific. In the phenotypically intermediate individuals different combinations of these species-specific bands were obtained. The patterns in these putative hybrids were not additive, which may be either the result of repeated backcrossing and introgression between the two species or of heterozygosity within the parental species. The results of the RAPD study are consistent with morphological analyses and allozyme data obtained for theGot-2 locus. Thus the RAPD markers used in this study may provide a powerful genetic tool for the identification of hybrids and the discrimination between the two pure species.     

The evolutionary significance of multigermicity in the genusSpinacia (Chenopodiaceae)

The presence of multigerm seedballs in the chenopodiacious genusSpinacia is noted. In the wild, colonising, and weedy dioecious species,S. turkestanica andS. tetrandra, the distribution of a multigerm seedball could effectively overcome the problem posed by isolation of the sexes. The hypothesis is tested assessing the extent of intra-seedball progeny hybridisations and seed production in the two wild species. The success in seed production by intra-seedball progeny crosses suggests that the distribution of such seedball progeny groups permits not only a percentage survival under isolation of these dioecious plants, but also the colonisation of areas outside that of the parent populations.     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.     

Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization

This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 mol m^–2 s^–1 after in vitro phases at 50 mol m^–2 s^–1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 mol m^–2 s^–1. The results taken as a whole suggest that 300 mol m^–2 s^–1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

Subtle behavioral variation in wild chimpanzees, with special reference to Imanishi’s concept of kaluchua

Here we consider the concept of kaluchua (a word adopted from the English culture) in group-living animals developed by Imanishi in the 1950s. He distinguished it from bunka (the Japanese equivalent to the English culture) because he thought that bunka had strong connotations of noble and intellectual human-like activities. Although he did not rigidly define kaluchua, his original concept of kaluchua was much broader than bunka and represented non-hereditary, acquired behavior that was acknowledged socially. However, instead of social life, complex feeding skills have often formed the central topic in the current studies of animal culture. In order to provide evidence that more subtle behavioral variations exist among wild chimpanzee (Pan troglodytes) populations, we directly compared the behaviors of two well-habituated chimpanzee groups, at Bossou and Mahale. During a 2-month stay at Bossou, M.N. (the first author) saw several behavioral patterns that were absent or rare at Mahale. Two of them, mutual genital touch and heel tap were probably customary for mature females and for mature males, respectively. Index to palm and sputter are still open to question. These subtle patterns occurred more often than tool use during the study period, suggesting that rarity is not the main reason for their being ignored. Unlike tool use, some cultural behavioral patterns do not seem to require complex skills or intellectual processes, and sometimes it is hard to explain the existence of such behaviors only in terms of function.     

Acclimation of the Sea Cucumber Apostichopus japonicus to Decreased Salinity at the Blastula and Gastrula Stages: Its Effect on the Desalination Resistance of Larvae at Subsequent Stages of Development

Apostichopus (= Stichopus) japonicus blastulae and gastrulae were acclimated for 18 h to salinities of 32 (control), 24 and 22 (the lower limit of the range of tolerance), and 20 (below the range of tolerance). Acclimation to 20 resulted in the appearance of teratic larvae, most of which subsequently died. Acclimation to 24, 22, and 20 led to a shift in the range of tolerance of the larvae at further stages of development. With a decrease in salinity, acclimated larvae developed more successfully than unacclimated larvae. Acclimated larvae attained the pentactula stage and settled at a salinity range of 32–20; unacclimated larvae, at 32–22. At different stages of development, acclimated larvae survived greater decreases in salinity than unacclimated larvae. The acclimation effects could be traced up to metamorphosis and settling, i.e., two weeks after the end of the acclimation process.     

Production of β-fructofuranosidase byAspergillus japonicus

A newly isolated strain, MU-2, which produces very high -fructofuranosidase activity, was identified asAspergillus japonicus. For enzyme production by the strain, sucrose at 20% (w/v) was the best carbon source and yeast extract at 1.5 to 3% (w/v) the best nitrogen source. Total enzymatic activity and cell growth were at maximum after 48 h, at 1.57×10⁴ U/flask and 0.81 g dry cells/flask, respectively. The optimum pH value of the enzymatic reaction was between 5.0 and 5.5 and the optimum temperature 60 to 65°C. The enzyme produced 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose by fructosyl-transferring activity. The strain was found to be very useful for industrial production of -fructofuranosidase.     

Differential tolerance of high manganese among rapeseed genotypes

Cultivation of crop cultivars resistant to high soil manganese (Mn) may reduce the negative effects of Mn toxicity on crop yield. Three studies were carried out to select Brassica genotypes (B. napus and B. rapa) resistant to high Mn concentration and to characterise the nature of any Mn resistance found. In Experiment 1, 33 B. napus and nine B. rapa genotypes were screened in a sub-irrigated nutrient solution system. Based on visual symptoms and plant size, single plants were identified with resistance to high Mn from within cultivars of four B. napus and one B. rapa. Resistance was also identified in one B. napus doubled haploid genotype. In Experiment 2, a genotype resistant to high Mn and two genotypes (progenies from Experiment 1) sensitive to high Mn were exposed to eight Mn concentrations (9–500 M) for 2 weeks in nutrient solution. The relative shoot weight (RSW) and the relative root weight (RRW) of the genotype resistant to Mn were significantly greater at 100 M Mn than both genotypes sensitive to high Mn; the sensitive genotypes reacted similarly. The three genotypes had similar tissue Mn contents and the elevated Mn tissue contents did not induce deficiencies of Mg or Fe. In Experiment 3, 12 genotypes (progenies from Experiment 1) were screened in nutrient solution at 9 M Mn and with an additional 125 M Mn. The RRW and RSW of the genotypes ranged from 35 to 114 and 39 to 94%, respectively. All the selections sensitive to high Mn had a RSW <60% and thus were confirmed to be Mn sensitive, while all the selections resistant to Mn had a RSW >70% and thus were confirmed as Mn resistant. This evidence confirmed the availability of rapeseed germplasm resistant to Mn toxicity with an ability to withstand high content of Mn through internal tissue tolerance. Also, the observed Mn tolerance in this material is genetically controlled and not an artifact of our screening assays.     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy

Summary We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/g DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/gm DNA).     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.