Oligosaccharide Specificity of the Fucolectin from the Bark of Laburnum (Laburnum anagyroides)

A comparative study of fine carbohydrate specificity of the lectin from the bark of laburnum Laburnum anagyroides (LABA) and the fucolectin from asparagus pea Tetragonolobus purpureus (TPA) was performed using inhibition of agglutination of the complex formed by H-active neoglycoprotein and nanoparticles of colloidal gold. Both lectins bound most strongly the H type 2 oligosaccharides comprising O-glycans; however, LABA was almost unable to discriminate between them. LABA bound more weakly the H type 6 trisaccharide (Fuc1-2Gal1-4Glc) and difucosyllactose (Fuc1-2Gal1-4[Fuc1-3]Glc), a glucoanalogue of the Le^y antigen, and, even more weakly, the Le^a pentasaccharide lacto-N-fucopentaose II (Gall-3[Fucl-4]GlcNAcl-3Gall-4Glc). However, LABA did not bind the antigens Le^b, Le^c, and Le^d, very poorly interacted with the terminal Le^x, and somewhat more strongly bound the internal Le^x. The lectin also had a hydrophobic binding site. Both lectins exhibited a cluster effect with polymeric ligands (neoglycoproteins).     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline     

Structural and immunochemical identification of Leb glycolipids in the plasma of a group O Le(a-b-) secretor

Total non-acid glycosphingolipids were isolated from the plasma of a healthy red blood cell group O Le(a-b-) salivary ABH secretor individual. Glycolipids were fractionated by HPLC and combined into eight fractions based on chromatographic and immunoreactive properties. These glycolipid fractions were analysed by thin-layer chromatography and tested for Lewis activity with antibodies reactive to the type 1 precursor (Le^c), H type 1 (Le^d), Le^a and Le^b epitopes. Fractions were structurally characterized by mass spectrometry (EI-MS and LSIMS) and proton NMR spectroscopy. Expected blood group glycolipids, such as H type 1, (Fuc1-2Gal1-3GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified. Inconsistent with the red cell phenotype and for the first time, small quantities of Le^b blood group glycolipids (Fuc1-2Gal1-3(Fuc1-4)GlcNAc1-3Gal1-4Glc1-1Cer) were immunochemically and structurally identified in the plasma of a Lewis-negative individual. These findings confirm recent immunological evidence suggesting the production of small amounts of Lewis antigens by Lewis negative individuals. Abbreviations: HPLC, high performance liquid chromatography; TLC, (high performance) thin layer chromatography; EI-MS, electron impact ionisation mass spectrometry; LSIMS, liquid secondary ion mass spectrometry; NMR, nuclear magnetic resonance spectroscopy. The sugar types are abbreviated to Hex for hexose, HexNAc forN-acetylhexosamine and dHex for deoxyhexose (fucose). The ceramide types are abbreviated to d for dihydroxy and t for trihydroxy base, n for non-hydroxy and h for hydroxy fatty acids; LCB, long chain base.     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.     

Characterization of quantitative trait loci (QTLs) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani)

Sheath blight, caused by Rhizoctonia solani, is one of the most important diseases of rice. Despite extensive searches of the rice germ plasm, the major gene(s) which give complete resistance to the fungus have not been identified. However, there is much variation in quantitatively inherited resistance to R. solani, and this type of resistance can offer adequate protection against the pathogen under field conditions. Using 255 F₄ bulked populations from a cross between the susceptible variety Lemont and the resistant variety Teqing, 2 years of field disease evaluation and 113 well-distributed RFLP markers, we identified six quantitative trait loci (QTLs) contributing to resistance to R. solani. These QTLs are located on 6 of the 12 rice chromosomes and collectively explain approximately 60% of the genotypic variation or 47% of the phenotypic variation in the LemontxTeqing cross. One of these resistance QTLs (QSbr4a), which accounted for 6% of the genotypic variation in resistance to R. solani, appeared to be independent of associated morphological traits. The remaining five putative resistance loci (QSbr2a, QSbr3a, QSbr8a, QSbr9a and QSbr12a) all mapped to chromosomal regions also associated with increased plant height, three of which were also associated with QTLs causing later heading. This was consistent with the observation that heading date and plant height accounted for 47% of the genotypic variation in resistance to R. solani in this population. There were also weak associations between resistance to R. solani and leaf width, which were likely due to linkage with a QTL for this trait rather than to a physiological relationship.     

Red cell H-deficient,salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin

Based on the genetic model proposing thatH andSe are two structural genes, we predicted that the red cell H-deficient, salivary ABH secretor phenotype should be found on Reunion island, where a large series of H-deficient non-secretor families have been previously described. Two such Reunion individuals are now reported. POU [A_h, Le(a–b+), secretor of A, H, Le^a and Le^b in saliva] and SOU [O_h, Le(a–b+), secretor of H, Le^a and Le^b in saliva]. Both are devoid of H -2-fucosyltransferase activity in serum. In addition, the preparation of total non-acid glycosphingolipids from plasma and red cells of POU revealed the type 1ALe^b heptaglycosylceramide and small amounts of the monofucosylated type 1 A hexaglycosylceramide. Both glycolipids possess an H structure probably synthesised by the product of theSe gene. No other blood group A glycolipids, with types 2, 3 or 4 chains, normally present in the presence of the product of theH gene, were found on red cells or plasma of POU.TheH,Se andLe genetic control of the expression of ABH and related antigens in different tissue structures of the skin is described in 54 H-normal individuals of known ABO, secretor and Lewis phenotypes; in one red cell H-deficient salivary secretor (SOU); and in one H-deficient non-secretor (FRA). Sweat glands express ABH under the control of theSe gene. Sweat ducts express ABH under the control of bothH andSe genes and Lewis antigens under the control ofLe and bothH andSe genes. Epidermis, vascular endothelium and red cells express ABH under the control of theH gene. The products ofH andSe genes are usually expressed in different cells. However, the results illustrate that in some structures, like the epithelial cells of sweat ducts, both the products ofH andSe genes can contribute to the synthesis of the same Le^b structure.     

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [-D-Gal-(14)-D-Glc], N-acetyllactosamine [-D-Gal-(14)-D-GlcNAc] and lacto-N-biose [-D-Gal-(13)-D-GlcNAc].     

Characterization of theGeosiphon pyriforme symbiosome by affinity techniques: confocal laser scanning microscopy (CLSM) and electron microscopy

Summary Geosiphon pyriforme represents a photoautotrophic endosymbiosis of aGlomus-like fungus with the cyanobacteriumNostoc punctiforme. The fungus forms unicellular bladders of up to 2 mm in length and 0.5 mm in diameter growing on the soil surface and harboring the endosymbioticNostoc filaments. The cyanobacteria are located in a compartment (the symbiosome) bordered by a host membrane. The space between this symbiosome membrane (SM) and theNostoc cell wall is filled with an about 30–40 nm thick layer of amorphous material, which is present also in the regions of the symbiosome where noNostoc filaments are located. At these sites the amorphous material consists of a 20–30 nm thick layer separating the SM. The region between the SM and the cyanobacterium is defined as symbiosome space (SS). Fungal bladders, hyphae and free livingNostoc were analyzed by affinity techniques as well as the material occurring in the SS. FITC-coupled lectins with sugar specificity to -D-mannosyl/-D-glucosyl (Con A), N-acetyl--D-glucosamine oligomers (WGA), -L-fucosyl (UEA-I), -D-galactosyl (RCA-120), -D-galactosyl (BS-I-B₄), N-acetyl--D-galactosamine (HPA), and sialic acid (EBL) residues were tested. WGA binding and calcofluor white staining demonstrated that the bladder wall as well as the SS contain fibrillar chitin. Of the other lectins only Con A clearly labeled the symbiosome. On the contrary, the lectin binding properties of the slime produced by free livingNostoc-colonies indicate the presence of mannose, fucose, GalNAc, sialic acid, and galactose, while chitin or GlucNAc-oligomers could not be detected. The symbiosome was also investigated electron microscopically. WGA-gold binding confirmed the presence of chitin, while a slight PATAg reaction indicated some polysaccharidic molecules within the SS. Our results show that the amorphous material within the SS contains molecules typical of the fungal cell wall and suggest that the SM is related to the fungal plasma membrane. The applied lectins all bind to the hyphal surface, indicating a high molecular complexity. Mannosyl, -galactosyl, and sialic acid residues are strongly exposed at the outer cell wall layer, whereas GlucNAc, GalNAc, and -galactosyl residues seem to be present in smaller amounts. The symbiotic interface established between the fungus andNostoc inGeosiphon shows many similarities to that occurring between fungi and root cells in arbuscular mycorrhizas.Abbreviations AM arbuscular mycorrhiza - BS-I-B₄ Bandeiraea simplicifolia lectin I isolectin B₄ - CLSM confocal laser scanning microscopy - Con A Concanavalin A - EBL elderberry bark lectin I - FITC fluorescein isothiocyanate - HPA Helix pomatia agglutinin - PATAg periodic acid-thiocarbohydrazide-Ag proteinate - SM symbiosome membrane - SS symbiosome space - RCA-120 Ricinus communis agglutinin 120 - UEA-I Ulex europaeus agglutinin I - WGA wheat germ agglutinin Dedicated to Professor Dr. Peter Sitte at the occasion of his 65th birthday     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

A novel wheat alpha-amylase gene (alpha-Amy3)

Summary A genomic clone of a wheat -amylase gene (Amy3/33) was identified, on the basis of hybridisation properties, as different from -Amy1 and -Amy2 genes which had been characterised previously. The nucleotide sequence revealed that this gene has the normal sequence motifs of an active gene and an open reading frame interrupted by two introns. The protein sequence encoded by this open reading frame is recognisably similar to that of -amylase from the -Amy1 and -Amy2 genes and there is high sequence homology in all three proteins at the putative active sites and Ca⁺⁺ binding region. In addition, the introns are at positions equivalent to the position of introns in the -Amy1 and -Amy2 genes. However, the sequence was less similar to -Amy1 and -Amy2 than these are to each other. Southern blot analysis showed that the Amy3/33 DNA is one of a small multigene family carried on a different chromosome (group 5) from either the -Amy1 or -Amy2 genes. A further difference from the -Amy1 and -Amy2 genes was the pattern of expression. Amy3/33 was expressed only in immature grains and, unlike the -Amy1 and -Amy2 genes, not at all in germinating aleurones. These data suggested therefore that this gene represents a third type of -amylase gene, not described before, which shares a common evolutionary ancestor with the -Amy1 and -Amy2 genes.     

Fruiting studies in species ofCapronia (Herpotrichiellaceae)

A recently described protocol for thein vitro production of ascomata was employed to determine the sexual incompatibility systems of five species ofCapronia. The formation of mature ascomata in isolates derived from single ascospores demonstrated thatC. epimyces, C. mansonii, andC. munkii n. sp. are homothallic. In contrast, fertile ascomata were observed only in mass-ascospore isolates and pairwise crosses between specific single-ascospore isolates inC. dactylotricha n. sp. andC. moravica. TheExophiala anamorphs ofC. dactylotricha andC. munkii are described and aPhialophora-like synanamorph is reported for the former species. Germinating ascospores ofC. munkii formed conidiogenous cells directly, while the ascospores of the remaining species germinated to produce germ tubes and hyphae. The application of the terms microcyclic conidiation to secondary conidium production and sclerotial body and stroma to the multicellular structures produced by species ofCapronia andExophiala are discussed.