Experimental documentation of natural hybridization inCactaceae: Origin of Lloyd's Hedgehog Cactus,Echinocereus ×lloydii

The origin ofEchinocereus ×lloydii Britt. & Rose, pro sp. (Lloyd's Hedgehog Cactus) was investigated using comparative morphology, cytology, biochemistry, and particularly, artificial hybridization. Numerous artificial crosses between the putative parentsE. coccineus Engelm. (a species of claret-up cactus) andE. dasyacanthus Engelm. (Texas Rainbow Cactus) were successful, resulting in the production of hundreds of seeds with hybrid embryos. The F₁ hybrid progeny (i.e., syntheticE. ×lloydii) grew to sexual maturity in about four and one-half years, whereupon successful backcrosses and F₂ generation hybrids were also obtained. The known F₁ hybrids closely approximated naturalE. ×lloydii. The fertility of these syntheticE. ×lloydii was high, like their natural counterparts. The populations ofE. ×lloydii in Pecos County, Texas are inferred to have originated as the result of natural interspecific hybridization. It is assumed thatE. ×lloydii or similar plants may arise wherever the parental taxa grow sympatrically.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Neue Taxa vonTillandsia subgenusDiaphoranthema (Bromeliaceae) aus Bolivien und Argentinien

5 new taxa are described and illustrated; their position within the subgenus is discussed:T. hirta W. Till & L. Hromadnik,T. cotagaitensis L. Hromadnik,T. caliginosa W. Till, andT. gilliesii Baker subsp.polysticha W. Till & L. Hromadnik are members of a group includingT. myosura Grisebach exBaker,T. mandonii E. Morren exMez in DC.,T. retorta Grisebach exBaker em.Grisebach andT. andicola Gillies exBaker. T. brealitoensis L. Hromadnik is related toT. angulosa Mez in DC. but distinct and possibly of hybrid origin.

     

Electrophoretic confirmation of the intergeneric hybrid ×Ruttyruspolia (Acanthaceae)

A plant collected in South Africa in the early 1960's has been considered an intergeneric hybrid with the parental taxa beingRuspolia hypocrateriformis (Vahl)Milne-Redhead var.australis Milne-Redhead andRuttya ovata Harv. The intermediate morphology of the plant provided the strongest evidence of its hybrid origin. The natural hybrid, named formally as ×Ruttyruspolia A. Meeuse & de Wet, is highly sterile. Crosses between the two presumed parental taxa produced two plants that are very similar to the putative natural hybrid. We had examined the presumed parental species and the natural and artificial hybrids using enzyme electrophoresis. The two parental species are highly differentiated at genes specifying soluble enzymes; they have a genetic identity of 0.51. They have no common alleles at two genes, and contain alternative alleles in very different frequencies at two loci.Ruttya andRuspolia exhibit both unique and common alleles at two additional genes. The natural and artificially produced plants of ×Ruttyruspolia are identical electrophoretically and contain alleles unique to each of the parental species at two genes. In addition, individuals of ×Ruttyruspolia combine alternative high frequency alleles from each parent at two loci. Allozymes provide strong confirming evidence for the hybrid origin of naturally occurring ×Ruttyruspolia because the products of specific alleles either unique to or highly characteristic of the two putative parental taxa are found combined in ×Ruttyruspolia.     

Biometrische Untersuchungen an Populationen vonOphrys cornuta,O. holosericea und ihren Hybriden (Orchidaceae)

Population variability ofOphrys holosericea (Burm. f.)Greut. subsp.holosericea (=O. fuciflora Crantz subsp.fuciflora) from near Vienna (Austria), and of subsp.maxima (Fleischm.)Greut. andO. cornuta Steven with intermediates from the Dalmatian island Hvar (Yugoslavia) was analysed and illustrated by scatter diagrams. A hybrid origin of these intermediates is suggested. Aspects of hybridization betweenO. holosericea agg. andO. scolopax agg. are discussed.

     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Ribosomal RNA sequence comparisons demonstrate an evolutionary relationship betweenZygnematales and charophytes

The nuclear-encoded small subunit ribosomal RNA (rRNA) sequences were determined forGenicularia spirotaenia, Mesotaenium caldariorum, andStaurastrum spec. (Zygnematales) to elucidate the evolutionary position of these green algae. Results of neighbour-joining and maximum parsimony phylogenetic analyses support a monophyletic origin of theZygnematales within the evolutionary assemblage defined by theCharophyceae (sensuMattox & Stewart) and land plants. TheZygnematales/Charophyceae/land plants are evolutionarily distinct from the monophyletic lineage defined by theChlorodendrales, Pseudoscourfieldiales, and theMicrothamniales/Chlorophyceae. In memoriamRobert W. Hoshaw.     

Cytotaxonomische Untersuchungen in der Artengruppe desRanunculus alpestris (Ranunculaceae)

Morphological and cytological investigations as well as crossing experiments were carried out with the 5 species of theRanunculus alpestris group (R. alpestris L.,R. traunfellneri Hoppe,R. bilobus Bertol.,R. crenatus Waldst. etKit.,R. magellensis Ten.). A key to the species is presented; localities and distribution are given in addition to extensive diagnoses. Crossing experiments between the 5 taxa were successful (F₁-F₃ individuals, backcross types, tripeland quadrupelbastards); the morphology of the experimentally obtained F₁-hybrids was mostly intermediate. All 5 species as well as all hybrids have a chromosome number of 2n = 16; there is no statistically significant difference between the karyotypes of the 5 taxa. According to the results of the morphological investigations and the crossing experiments we can distinguish 2 subunits of very closely related species: a)R. alpestris, R. traunfellneri, R. bilobus; b)R. crenatus, R. magellensis. The speciation within the group ofRanunculus alpestris is discussed.

     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

A comparative electrophoretic study of the seed albumins fromSophora microphylla andPisum sativum (Leguminosae)

The albumin proteins from seed ofSophora microphylla Ait. and from cotyledons ofPisum sativum L. (cv. Greenfeast) have been analysed electrophoretically using a range of gels of varied pore size. Plots of mobility [as 100 log₁₀ (R _f × 100)] vs.acrylamide content of gel indicate that very few of the albumins fromS. microphylla are homologous with albumins fromP. sativum. Despite the diverse compositions of the two fractions, their amino acid analyses were surprisingly similar.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Biosynthesis of methyl chloride in the fungusPhellinus pomaceus

The biosynthetic origin of themethyl group in the methyl chloride produced by cultures ofPhellinus pomaceus (Pers.) Maire has been investigated using stable isotope labeled substrates. Feeding ofd-[6,6-²H₂] glucose,Dl-[3,3-²H₂] serine andl-[methyl-²H₃] methionine led to the production of deuterated methyl chloride in which the major labeled species contained 2, 2, and 3 deuterium atoms, respectively. The data are consistent with the methyl chloride produced by this organism being derived solely from methionine with retention of all of the methyl protons.Abbreviation SAM S-adenosylmethionine - FH₄ tetrahydrofolate - N⁵-CH₃FH₄ N⁵-methyltetrahydrofolate - B₁₂ cobalamin     

Cyclamen (Primulaceae) in tropical Africa

Cyclamen somalense Thulin & Warfa, spec. nova, the first member of the genus known from tropical Africa, is described from the Al Miskat Mts in NE. Somalia. The new species is closely related to the E. MediterraneanC. persicum Mill. The disjunct Mediterranean element in the mountain flora of northern Somalia, to whichC. somalense belongs, is believed to be largely a relict of Tertiary origin.     

Morphological and cytological evidence for polyphyletic allopolyploidy inArctostaphylos mewukka (Ericaceae)

The central Sierran tetraploidArctostaphylos mewukka Merriam has been reported to be an allopolyploid originating from the diploid species,A. patula Greene and eitherA. viscida subsp.viscida Parry orA. viscida subsp.mariposa (Dudley)P. V. Wells, although without conclusive evidence. Morphometrics and the verification and determination of chromosome numbers were used to substantiate the evolutionary relationships among these species. A closely related species,Arctostaphylos truei Knight, was also examined using these methods to determine its separability fromA. mewukka. The morphometric analyses support a hypothesis for a polyphyletic origin ofA. mewukka from different races ofA. viscida andA. patula. The chromosomal data, although inclusive, also support this hypothesis. The data do not support the recognition ofA. truei as a taxonomic entity separate fromA. mewukka at the species level.     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

Chemical composition of Eubacterium alactolyticum cell wall peptidoglycan

The mechanism of lysis of Eubacterium alactolyticum cell walls by Streptomyces albus G enzyme was studied. The analysis of the peptide terminal groups and peptide subunits isolated from the cell wall digest, released during solubilization of the cell walls, revealed that lytic action of S. albus G enzyme was mainly due to D-alanyl-A₂pm endopeptidase, N-acetylmuramyl-L-alanine amidase, N-acetylmuramidase and N-acetylglucosaminidase. E. alactolyticum cell wall peptidoglycan is composed mainly of glucosamine, muramic acid, D-glutamic acid, L- and D-alanine, meso-diaminopimelic acid and glycine. The peptide subunit consists of L-alanyl-D-glutamyl-meso-A₂pm-D-alanine. D-Alanine is connected directly with the amino group of the meso-A₂pm residue of another peptide subunit. All of the L-amino groups of meso-diaminopimelic acid are involved in cross-linking.The possible structure of the peptide moiety of E. alactolyticum cell wall peptidoglycan is presented.     

The genusPicris (Asteraceae,Lactuceae) in Tropical Africa

Three species of the genusPicris L. are native in Tropical Africa:P. abyssinica Sch. Bip. (Ethiopia),P. xylopoda Lack, spec. nova (Nigeria, Ethiopia) andP. humilis DC. (Senegal, Mali). There are indications that the two perennial species,P. abyssinica andP. xylopoda, are related to and have evolved from a primitive Central Asiatic stock in a manner parallel to many African species ofCrepis L.P. humilis, on the other hand, is a small annual plant with a high number of derived characters. The introduced species of European origin growing south of the Sahara are briefly mentioned.     

Genetic relationships between European and Siberian larch,Larix spp. (Pinaceae), studied by allozymes. Is the Polish larch a hybrid between these two species?

Thirteen populations ofLarix decidua subsp.decidua and subsp.polonica, and three populations ofL. sibirica were investigated by starch-gel electrophoresis. In the populations assayed 61 alleles at 17 loci were revealed. The allozyme data support the earlier observations about close relationships between these two larch species. Nei's genetic distances betweenL. decidua andL. sibirica were relatively small (D = 0.057), however, almost five times larger, on average, than those between populations of the same species. Results obtained in this study disagree withBobrov's hypothesis about the hybrid origin of the Polish larch and suggest a direct origin from the European larch.