A new enzymatic route to the synthesis of 12-ketoursodeoxycholic acid

Summary Cholic acid (3,7,12-trihydroxy-5-cholanoic acid) was completely and selectively transformed into 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid) by means of two consecutive enzymatic steps catalyzed, the first, by 7- and 12-hydroxysteroid dehydrogenase and, the second, by 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with -ketoglutarate-glutamate dehydrogenase and glucose-glucose dehydrogenase, respectively.     

Donor tissue and culture condition effects on mesophyll protoplasts of Medicago sativa

Mesophyll protoplasts were produced from clones of two cultivars of Medicago sativa, Rangelander and Regen S. Protoplasts from the Regen S clone generally gave rise to calli while those from the Rangelander clone would undergo direct embryogenesis. Effects of plant growth conditions, donor tissue pretreatment and protoplast culture conditions on mesophyll protoplast production and subsequent development patterns were investigated. The major factor determining whether or not mesophyll protoplasts would be produced from either of the clones was the pretreatment in water of shoots excised from the donor plants. Pretreatment in water containing growth regulators did not alter protoplast production or development in the Regen S clone. Pretreatment of the Rangelander clone shoots with abscisic acid or naphthaleneacetic acid was slightly beneficial to embryo production while pretreatment with benzylaminopurine was detrimental. Altered leaf morphology induced by growth condition changes did not affect mesophyll protoplast production or subsequent development patterns when shoots were pretreated in water. Culture of protoplasts in liquid droplets or solid agar medium increased low density protoplast survival and subsequent embryo production in the Rangelander clone.     

Production of thermostable amylolytic enzymes by Thermococcus hydrothermalis

A cell extract of Thermococcus hydrothermalis, grown for 6 h, gave -glucosidase activity at 14.9 U/l, degrading oligosaccharides and maltose. -Amylase, -glucosidase and pullulanase activities were detected at 289 U/l, 13.5 U/l and 30 U/l respectively in the culture medium after 24 h growth of the archaeum. All of three enzymes, characterised by a half-life time of 1 to 5 h at 95°C, degraded both the (14) and (16) linkages of polysaccharides and the (14) linkages of oligosaccharides. © Rapid Science Ltd. 1998     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased     

Carotenoid pigments of Sorangium compositum (Myxobacterales) including two new carotenoid glucoside esters and two new carotenoid rhamnosides

Summary The carotenoid pigments of the myxobacterium Sorangium compositum were analyzed by chromatographical and chemical techniques and by visible, infra red, and mass spectroscopy. Besides -carotene, neurosporene, torulene, lycopene, and 1,2-dihydro-1-hydroxy--carotene, four new carotenoid glycosides were found. These pigments were identified as 1,2-dihydro-1-hydroxy-torulene glucoside ester (I), 1,2-dihydro-3,1-dihydroxy-torulene glucoside ester (III), 1,2-dihydro-1-hydroxy-torulene rhamnoside (II), and 1,2-dihydro-3,1-dihydroxytorulene rhamnoside (IV).Fifth communication on the carotenoids of myxobacteria. Fourth communication see Arch. Mikrobiol. 76, 364–380 (1971).     

Heterogeneous binding and killing behaviour of human γ/δ-TCR+ lymphokine-activated killer cells against K562 and Daudi cells

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, /-TCR) was compared in both fractions. Only LAK cells expressing the / T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that /-TCR⁺ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether /-TCR⁺ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of /-TCR⁺ cells were established. Against K562 cells, /-TCR⁺-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of /-TCR⁺ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of /-TCR⁺ LAK cells with anti-/ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that /-TCR⁺ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the /-TCR. Occupation of the /-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in /-TCR⁺ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.This work was supported by the Hartmann-Müller Foundation, Zürich     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work     

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum υ chain deduced from cDNA sequence

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C) chain. C is 433 amino acids long and organized into four domains (C1–C4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C is most closely related to Xenopus C (40% identical amino acid residues) and C1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C1 and C2 domains is consistent with an additional intra-domain S-S bond similar to that suggested for Xenopus C and C, and for the avian C and the human C. C4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C3, human C4, and avian and anuran C4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian chains, and mammalian chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number X69492. Correspondence to: J. S. Fellah.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

Organogenesis from ‘Passe Crassane’ and ‘Old Home’ pear (Pyrus communis L.) protoplasts and isoenzymatic trueness-to-type of the regenerated plants

Summary Large numbers of highly viable mesophyll protoplasts were isolated from shoot cultures of the scion cv Passe Crassane and the rootstock genotype Old Home of common pear (Pyrus communis L.). Protoplasts were cultured for both genotypes either as liquid layers or as liquid-over-agar cultures, in ammonium-free MS medium with 0.5 M mannitol, 50 mg/l casein enzymatic hydrolysate (CEH), 2.0 mg/l NAA and 1.0 mg/l BAP, plus either 0.5 mg/l IAA (for Old Home) or 2.0 mg/l IAA (for Passe Crassane). Protoplast microcalli, obtained by day 60 (Passe Crassane) or day 80 (Old Home), were transferred for further growth to ammonium-free MS medium with 2.0 mg/l NAA and 1.0 mg/l BAP. Shoot bud regeneration from the protoplastderived callus was first attempted between 100 (Passe Crassane) and 120 (Old Home) days after protoplast isolation. For Passe Crassane, shoot buds were regenerated (day 130) on a half-strength MS medium with 0.1 mg/l IBA, 0.5 mg/l BAP, 50 mg/l CEH and 20 mg/l Ca-panthotenate. For Old Home, shoot but regeneration only occurred 30 days later and on the same medium as above, which was additionally supplemented with double the concentration of the group B vitamins found in the original MS formulation and 0.05 mg/l GA₃. Following micropropagation and in vitro rooting of shoots, the plants were transferred to soil following standard procedures. Trueness-to-type of the regenerated plants was assessed by analysing their leaf isozyme banding profiles (for EST, AP, PRX, SOD, ENP, LAP, PGI, AAT, ADH, MDH and PGM) and comparing them to those corresponding to the original shoots that provided the protoplasts. No differences between the mother shoots and the protoclones were observed for any one of the 11 isozyme systems studied.     

Theoretical studies on the necessary number of components in mixtures

Summary Theoretical studies on the necessary numbers of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to risk considerations — using the binomial distribution and the Polya-distribution. The risk r of a mixture has been defined as the probability of catastrophic losses (catastrophe = decrease of productivity of q% or more by susceptibilities of the components). Using 1) the binomial distribution and 2) its generalization, the Polya-distribution, and several simplifying assumptions, the risks r = r (x, a, q, n) have been calculated numerically (n = number of components in the mixture, a = parameter for the intensity of contagion and dispersion of susceptibilities (for example: diseases and epidemics), x = probability of susceptibility). The Polya-model reduces to the binomial case if a = 0. The main results are: 1. For each number n of components the risk r decreases markedly with decreasing x (for each q and for each a). 2. For x < = q the risk r decreases with an increasing number n of components (for each a). 3. For each number n of components and x and q with x < q the risk r increases with increasing a. 4. For given q, x and a the functions r = r(n) are asymptotic for larger numbers n of components with n > n^*. In spite of further increasing numbers of components in the mixture the risk remains almost constant. For all situations, where the risk decreases with increasing n these numbers n^*, therefore, can be considered as necessary numbers of components in mixtures, n^* depends on q, x and a. Nevertheless, a global and rough conclusion can be formulated: In many situations one obtains necessary numbers of 30–40 components for a0 and 20–30 components for a = 0.     

Facile preparation of optically pure [α-2H]-α-amino acids

Summary Racemic [-²H]--amino acids were prepared by heating the corresponding amino acids (Phe, nor-Leu and Dopa) with 0.05 equivalents of benzaldehyde in deuterated-acetic acid. Based on¹H-nmr measurement, the isotopic purities of these racemized [-²H]--amino acids were found to be higher than 99.5%. Methylation of these isotope-labelled amino acids was achieved in methanol/thionyl chloride without affecting isotopic purity. Optically pure [-²H]--amino acids were obtained in high yield with high enantiomeric excess via alcalase catalysed resolution.     

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms viability and culturability are often equated. However, in recent years the apparently self-contradictory expression viable-but-nonculturable (VBNC) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms viability and culturability in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces VBNC with expressions that are internally consistent.     

Screening of turfgrass species and cultivars for NaCl tolerance

Summary Information regarding the relative levels of salt tolerance between cultivars of Kentucky bluegrass (Poa pratensis L.) is lacking. The objectives of this study were to 1) develop a simple, quick and sensitive method of screening turfgrass species for NaCl tolerance and 2) to compare the relative salt tolerance of five cultivars of Kentucky bluegrass (Ram I, Adelphi, Baron, Bensun, and Nassau) to other known salt tolerant turfgrass species such as alkalaigrass (Puccinellia distans (L.) Parl. cv. Fults) and two cultivars of red fescue (Festuca rubra L. Dawson, and Checker).Alkalaigrass and both cultivars of red fescue retained a high level of salt tolerance compared to the Kentucky bluegrass cultivars. Significant variability in salt tolerance was apparent among the Kentucky bluegrass cultivars with Adelphi and Ram I exhibiting the best overall tolerance.     

High-level Expression of Maize <Emphasis Type="Italic">γ</Emphasis>-zein Protein in Transgenic Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>)

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, Cabernet Sauvignon x Seyval and Chardonnay x Bianca, and a further six other grape genotypes (V. vinifera Sultanina, Merlot, Syrah, Müller-Thurgau, Vitis Regent and V. riparia Gloire de Montpellier). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross Chardonnay x Bianca. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d²) were calculated as a tool for predicting highly outbred recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.     

A method for the estimation of χ1 torsion angles in proteins

Summary A method for estimating CH-CH coupling constants from the shape and fine structure of NH-CH fingerprint-region cross peaks of COSY spectra is presented. Spectral simulations have been used to analyse the effect of variations in ³J_NH-CH, ³J_CH-CH, linewidths and digital resolution on the appearance of NH-CH COSY cross peaks. On the basis of these simulations a set of rules for broadly categorising experimental NH-CH cross peaks according to CH-CH coupling constants has been devised. The method has been applied to the analysis of NH-CH cross peaks of hen lysozyme. The results are compared to previous measurements of CH-CH coupling constants using E.COSY techniques.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.