Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy

Summary A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance ¹³C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.     

Evaluation of coupling conditions for the incorporation of Nα-Fmoc-Tyr(PO3H2)-OH in solid-phase peptide synthesis

Summary In order to minimise the formation of the pyrophosphate derivative of the target peptide when side-chain-unprotected phopshotyrosine is used in solid-phase peptide synthesis, this building block can be incorporated using benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate/1-hydroxybenzotriazole/N-methylmorpholine (1:1:2.3) in the presence of a chaotropic salt (0.4 M LiCl in N-methyl-2-pyrrolidinone).Abbreviations BOP benzotriazolyloxy-tris-(dimethylamino)phosphonium hexafluorophosphate - DIEA diisopropylethylamine - Fmoc 9-fluorenylmethoxycarbony - HATU N-[(dimethylamino)1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethan-aminium hexafluorophosphate N-oxide - HOBt 1-hydroxybenzotriazole - HPLC high-performance liquid chromatography - MALDI-TOF matrix-assisted laser-desorption ionization time-of-flight mass spectrometry - NMM N-methylmorpholine - NMP N-methyl-2-pyrrolidinone - Pmc 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl - ® solid support - TFA trifluoroacetic acid - TPTU 2-(2-pyridon-1-yl)-1,1,3,3-tetramethyluroniumfluoroborate. Abbreviations used for amino acids follow the recommendations of the IUPAC-IUB Commission of Biochemical Nomenclature [Eur. J. Biochem., 138 (1984) 9]     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

The use of arbuscular mycorrhizae to control onion white rot (Sclerotium cepivorum Berk.) under field conditions

 A field experiment was carried out to determine the effects of the inoculation of onion (Allium cepa L.) with Glomus sp. Zac-19 on the development of onion white rot (Sclerotium cepivorum Berk.) and on onion production. Mycorrhization delayed onion white rot epidemics by 2 weeks and provided a significant protection against the disease for 11 weeks after onion transplanting, as compared with nonmycorrhizal controls. Mycorrhizal plants showed an increase of 22% in yield, regardless of the presence of the white rot pathogen. Accepted: 8 January 1996     

A new method to determine the aw range in which immobilized lipases display optimum activity in organic media

Summary We describe a qualitative method to predict the pre-equilibration a_w, system value in which, covalent immobilized lipase B from Candida antarctica to sepharose and silica, displayed best synthetic activity. The methodology is based in the analysis of the water adsorption isotherms of the biocatalyst in air and in the organic solvent. The biocatalyst is active at pre-equilibration a_w values higher than the divergence point between both isotherms. In addition, native and immobilized lipase display highest activity if the biocatalyst is pre-equilibrated at a_w=P point. For preparative purposes, the validity of the method was proved in the esterification of racemic 2-(4-isobutyl phenyl) propionic acid with 1-propanol in isooctane at long reaction time.     

Sustainable ecotourism in Costa Rica: the Monteverde Cloud Forest Preserve

The paper discusses the sustainability of the Monteverde Cloud Forest Preserve in the context of Costa Rican ecotourism. While the history of the Preserve is somewhat unique, the analysis of visitation, financial, ecological and economic factors provides a convincing case that tourism at the Preserve is sustainable. The experience of the Preserve is also put in the context of Costa Rican ecotourism, particularly to the national parks. The paper concludes that the Preserve has played a very important role in the development of Costa Rica as an ecotourism destination. Nonetheless, the failure of experience at the Preserve to inform recent changes in national park pricing policy reveal that Costa Rica has yet to fully capitalize on the experience gained and lessons learned at the Preserve.     

EPR Spin Trapping Study of the Decomposition of Azo Compounds in Aqueous Solutions by Ultrasound: Potential for Use as Sonodynamic Sensitizers for Cell Killing

Sonodynamic therapy, a promising new approach to cancer treatment, is based on synergistic cell killing by combination of certain drugs (sonosensitizers) and ultrasound. Although the mechanism of sonodynamic action is not understood, the role of free radicals produced from sonosensitizers by ultrasound is implicated. In this work, we studied formation of free radicals during the decomposition of several water-soluble azo compounds by 50 kHz ultrasound in aqueous solutions. Using the spin trap 3, 5-dibromo-4-nitrosobenzene sulfonate (DBNBS) tertiary carbon-centered radicals from 2, 2'-azobis (N,N'-dimethyl-eneisobutyramidine) dihydrochloride (VA-044), 2-(carbamoylazo)-isobutyronitrile (V-30), and 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and CH3 radicals from 1, 1'-azobis (N,N'-dimethylformamide) (ADMF) were detected in argonsaturated solutions and the corresponding oxygen-centered radicals (alkoxyl and peroxyl) from VA-044, V-30, and AAPH were identified using the spin trap 5, 5'-dimethyl-l-pyrroline-N-oxide (DMPO) in aerated sonicated solutions. No free radicals from 4, 4'-dihydroxyazobenzene-3, 3'-dicarboxylic acid, disodium salt (DHAB) could be found in either system. While VA-044 and AAPH could also be readily decomposed by heat (42.5°C and 80°C), V-30 decomposition only occurred in the ultrasound-exposed solutions. The most likely mechanism of decomposition of azo compounds by ultrasound is their thermolysis in the heated shell of the liquid surrounding ca vita ting bubbles driven by ultrasound and/or by pyrolysis inside these bubbles. Experiments using scavengers of ·OH and ·H, which are produced by sonolysis in aqueous solutions, demonstrated that these radicals are not involved in the ultrasound-mediated radical production from the azo compounds. Due to the known cytotoxic potential of free radicals produced from azo compounds, the use of these compounds as ultrasound sensitizers appears to be a promising approach for sonodynamic cell killing.     

Karyological variation in the group ofMyosotis alpestris (boraginacea)

A total of 6 population samples ofMyosotis stenophylla Knaf, a rare species showing great ecological disjunction in its distribution, were examined to clarify the present status of its karyological variation. In order to elucidate relationships between lowland tetraploid populations ofM. stenophylla and diploid and tetraploid montane populations ofM. alpestris F.W. Schmidt, four population samples ofM. alpestris were also examined. The karyotypes of all populations ofM. alpestris s.l. studied were highly asymmetrical and heterogeneous, being composed of metacentric, submetacentric, subtelocentric and satellited acrocentric chromosomes. The karyotype formula for haploid chromosome set was established: n=x=12=6m+2sm+3st+1t^SAT. Multivariate analysis based on chromosome length and shape showed significant differences between diploid and tetraploid forms ofM. alpestris s.l. Four numerical parameters, used to characterize the karyotype ofM. stenophylla, revealed significant differences between populations on serpentine and on non-serpentine substrates. In addition, the noticeable affinity of the karyotype of non-serpentine populations to that ofM. alpestris tetraploids has been shown by means of discriminant analysis. These data suggest that the unique features of serpentine play an important role in the origin of karyotypic differentiation within populations ofM. stenophylla.