Comparison of the solution and crystal conformations of (G + C)-rich fragments of DNA.

DNA fragments crystallize in an unpredictable manner, and relationships between their crystal and solution conformations still are not known. We have studied, using circular dichroism spectroscopy, solution conformations of (G + C)-rich DNA fragments, the crystal structures of which were solved in the laboratory of one of the present authors. In aqueous trifluorethanol (TFE) solutions, all of the examined oligonucleotides adopted the same type of double helix as in the crystal. Specifically, the dodecamer d(CCCCCGCGGGGG) crystalized as A-DNA and isomerized into A-DNA at high TFE concentrations. On the other hand, the hexamer d(CCGCGG) crystallized in Z-form containing tilted base pairs, and high TFE concentrations cooperatively transformed it into the same Z-form as adopted by the RNA hexamer r(CGCGCG), although d(CCGCGG) could isomerize into Z-DNA in the NaCl + NiCl2) aqueous solution. The fragments crystallizing as B-DNA remained B-DNA, regardless of the solution conditions, unless they denatured or aggregated. Effects on the oligonucleotide conformation of 2-methyl-2,4-pentanediol and other crystallization agents were also studied. 2-Methyl-2,4-pentanediol induced the same conformational transitions as TFE but, in addition, caused an oligonucleotide condensation that was also promoted by the other crystallization agents. The present results indicate that the crystal double helices of DNA are stable in aqueous TFE rather than aqueous solution.     

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 . 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 . 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays). (c) 1996 John Wiley & Sons, Inc.     

Gram-scale purification of eicosapentaenoic acid (EPA, 20:5n-3) from wetPhaeodactylum tricornutum UTEX 640 biomass

Eicosapentaenoic acid (EPA, 20:5n-3) was obtained from the microalgaPhaeodactylum tricornutum following a three-step process: fatty acid extraction by direct saponification of wet biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds and EPA isolation by preparative HPLC. Direct saponification of wet biomass was carried out with KOH-ethanol (96% v:v) (1 h, 60 °C), extracting 91% of the EPA. PUFAs were concentrated by the urea method with an urea/fatty acid ratio of 4:1 at a crystallization temperature of 28 °C using methanol as the urea solvent. An EPA concentration ratio of 1.5 (55.2/36.3) and recovery of 79% were obtained. This PUFA concentrate was used to obtain 95.8% pure EPA by preparative HPLC, using a reverse-phase column (C₁₈, 4.7 cm i.d. × 30 cm) and methanol-water (1% AcH) 80:20 w/w as the mobile phase. Ninety-seven per cent of EPA loaded was recovered and 70% EPA present in theP. tricornutum biomass was recovered in a highly pure form by means of this three-step downstream processing. In each of the HPLC preparative runs, 635 mg PUFA concentrate were loaded, obtaining 326 mg of a highly concentrated EPA fraction (2.46 g d^–1). Finally, a preliminary cost statement has been calculated.     

Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Tetraploids inLuzula multiflora (Juncaceae) in Ireland: Karyology and meiotic behaviour

Populations ofLuzula multiflora s.l. in Ireland were examined karyologically. Plants from 14 populations were invariably tetraploid with 2n=24. Chromosomes of the tetraploid are of AL type (true tetraploidy). Meiosis of the tetraploids is of the same type as described for otherLuzula taxa in the literature. In meiosis, 12 bivalents are regularly formed. A hypothesis based on the morphological and allozyme data, that the tetraploids are of alloploid origin, is supported by the present results. Meiosis in an artificial hybrid between the presumed parental taxa,L. campestris andL. pallidula, was studied; a certain tendency towards chromosome doubling was observed. The geographical distribution of theL. multiflora cytotypes is also discussed.     

Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein.

Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2'',5''-phosphodiester bond (RNA branch) with the 5'' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.     

High elevation coniferous vegetation of Guatemala

A phytosociological study of the juniper (locally called huito), pine (locally called sacch), pine-alder and fir forests of the Sierra de los Cuchumatanes and Cadena Volcánica in Guatemala was carried out. The Zürich-Montpellier approach was followed. In total 119 relevés were sampled and the data were organised in phytosociological tables to distinguish vegetation clusters. TWINSPAN was used to evaluate major differences among plant communities. Seven zonal plant communities were distinguished and described, namely: (1)Relbunium microphyllum-Agrostis tolucensis, (2)Werneria nubigena-Agrostis exserta, (3)Lachemilla vulcanica-Pinus hartwegii, (4)Holodiscus argenteus-Pinus hartwegii, (5)Hypnum cypressiforme-Juniperus standleyi, (6)Agave hurteri-Alnus firmifolia and (7)Sabazia pinetorum-Abies guatemalensis. This paper provides a thorough floristic characterisation of each community and outlines the major anthropogenic activities. To conclude, ecologic and floristic (dis)similarities between plant communities of the study area and those of Central Mexico, like the different altitudinal distribution of fir forests and the establishment of mid-successional communities such as theAgave hurteri-Alnus firmifolia were discussed.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Keratinocyte growth factor

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex sterorid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.